Neutralization of CD95 activity blocks invasion of glioblastoma cells in vivo

ABSTRACT

The present invention relates to methods for treating an individual with high grade glioblastoma multiforme by preventing or disrupting the binding of CD95 to its ligand, CD95L, in vivo, whereupon that neutralization of CD95 activity reduces undesirable glial cell migration and invasion into body tissue.

CROSS REFERENCE TO RELATED APPLICATION

This application is divisional of U.S. Ser. No. 12/521,625, filed Sep.9, 2009, now U.S. Pat. No. 9,309,320; which is a 35 U.S.C. 371 NationalPhase Entry Application from PCT/EP2007/011461, filed Dec. 28, 2007,which claims the benefit of U.S. Provisional 60/877,367 filed on Dec.28, 2006, the disclosure of which are incorporated herein in theirentirety by reference.

FIELD OF THE INVENTION

The present invention relates to methods for treating an individual withhigh grade glioblastoma multiforme by preventing or disrupting thebinding of CD95 to its ligand, CD95L, in vivo, whereupon neutralizationof CD95 activity dramatically reduces the migration of cells invadingthe contralateral hemisphere.

BACKGROUND

Invasion of surrounding brain tissue by isolated tumor cells representsone of the main obstacles to an effective therapy of glioblastomamultiforme (GBM). Gliomas encompass the majority of tumors originatingin the central nervous system (CNS). In adults, the most common tumorsare high-grade neoplasms derived from astrocytes or oligodendrocytes.The World Health Organization classifies these malignant tumorsaccording to their degree of anaplasia into grade II (diffuseastrocytoma), grade III (anaplastic astrocytoma) and grade IV (GBM)¹.

Gliomas account for more than 50% of all brain tumors and are by far themost common primary brain tumors in adults. Despite, development of newdiagnostic technologies, the survival rate is extremely low. Only 3% arestill alive five years after diagnosis. The clinical outcome ofmalignant gliomas depends on the invasion of isolated tumor cells in thenormal brain tissue. Migrating cells can escape the surgical ablation ofthe tumor and are then the prime targets of post-surgical radiotherapyand adjuvant chemotherapy. Chemotherapeutic agents and irradiation actprimarily by inducing apoptosis. This induction of apoptosis ofteninvolves activation of the CD95 (Apo-1/Fas) death receptor/ligandsystem. Nevertheless, most malignant glioma cells are resistant toCD95-induced apoptosis. Here we show that triggering of CD95 increasesmigration/invasion in apoptosis-resistant human long-term and primaryglioma cultures. That is, triggering CD95 may entail initiating CD95activity by using an agonistic antibody to CD95 or recombinant CD95L.

The tendency of primary glioma tumors of migration over apoptosisincreases with the degree of malignancy. CD95 mediates migration via thePI3K/ILK/GSK3-beta; /MMP pathway in a caspase-independent manner.Furthermore we tried to figure out the linker molecule downstream ofCD95. A possible candidate was Phosphoprotein enriched inDiabetes/Phosphoprotein enriched in Astrocytes-15-kDalton” (PED/PEA-15).Knockdown experiments excluded PED/PEA-15 as linker molecule in thesignaling pathway of migration mediated through CD95/CD95L-System. Mostimportantly, gamma-irradiation also increased migration of cellsresistant to CD95-induced death. Irradiation-mediated migration could beblocked by neutralization of CD95L. Thus, a tumor's reaction to CD95stimulation should dictate subsequent therapy options. See Kleber, S.,“Gamma irradiation leads to CD95 dependent invasion in apoptosisresistant glioblastoma cells,” Ph.D. Thesis, DeutschesKrebsforschungszentrum, University of Heidelberg, Jan. 3, 2006(urn:nbn:de:bsz:16-opus-59926), which is incorporated herein byreference in its entirety.

The main types of gliomas are ependymomas, astrocytomas, andoligodendrogliomas, although there also exist mixed cellular forms ofglioma cell conditions, such as oligoastrocytomas.

In addition to a cellular characterization, gliomal tumors are alsocharacterized according to pathology and the seriousness of cellularinvasion, which is typically recognized by those in the field as a“grading” classification system.

The most commonly used grading system is the World Health Organization(WHO) grading system for astrocytomas. The WHO system assignsastrocytomas a grade from I to IV, with I being the least aggressive andIV being the most aggressive. Thus, pilocytic astrocytoma is an exampleof a WHO Grade I glioma; diffuse astrocytoma is an example of WHO GradeII; anaplastic (malignant) astrocytoma is an example of WHO Grade III;and glioblastoma multiforme is an example of WHO Grade IV. The latter isthe most common glioma in adults and, unfortunately, has the worstprognosis for inflicted patients.

Generically, the “low grade” gliomas are typically well-differentiated,slower growing, biologically less aggressive, and portend a betterprognosis for the patient; while the “high grade” gliomas, areundifferentiated or anaplastic, fast growing and can invade adjacenttissues, and carry a worse prognosis. High grade gliomas are highlyvascular tumors and have a tendency to infiltrate tissues, createnecrosis and hypoxia, and destroy the blood-brain barrier where thetumor is located.

There also are infratentorial gliomas, which occur mostly in childrenand supratentorial in adults. The infratentorial gliomas are located inall interior cerebral areas below the undersurface of the temporal andoccipital lobes, extending to the upper cervical cord, and includes thecerebellum. The supratentorial region is located above the tentoriumcerebelli and contains the forebrain.

Tumor grade is an important prognostic factor: median survival for gradeIII astrocytomas is 3 to 4 years and for grade IV astrocytomas 10 to 12months. The most frequent glioma (65%) is the GBM¹. Cellular resistanceto multiple proapoptotic stimuli and invasion of migrating tumor cellsinto the normal surrounding brain tissue are the main obstacles to aneffective therapy.

The current treatment of malignant gliomas (grade III and IV) involvessurgery, followed by irradiation and chemotherapy. Chemotherapeuticagents and γ-irradiation act primarily by inducing apoptosis. Inductionof apoptosis often involves activation of the CD95/CD95-ligand (CD95L;Apo1L/FasL) death system^(2,3). Binding of trimerized CD95L by the CD95receptor leads to recruitment of the adapter protein FADD(Fas-associated death domain, MORT1)⁴ and caspase-8 and -10 into adeath-inducing signaling complex (DISC)⁵. FADD contains a death domain(DD) and a death-effector domain (DED). Via its DD, FADD binds to the DDof CD95⁶. The DED recruits the DED-containing procaspase-8 into theDISC⁷. Procaspase-8 at the DISC is activated through self-cleavage andcommits the cell to apoptosis by activation of downstream effectorcaspases⁸.

The CD95/CD95L system is used by malignant glioma cells to increasetheir invasion capacity. CD95 induces cell invasion via thePI3K/ILK/GSK3β pathway and subsequent expression of metalloproteinases.Increased CD95L expression is exhibited by irradiated glioma cells thatescape surgical ablation. Irradiation-induced CD95 activity increasedglioma cell migration. In a murine syngenic model of intracranial GBM,CD95/CD95L expression was strikingly upregulated upon interaction withthe surrounding brain stroma.

The degree of CD95 and CD95L expression positively correlates with thetumor grade. Here we show that triggering of CD95 caused apoptosis inless malignant tumors (WHO I-II) while the grade IV tumors wereresistant to CD95-induced apoptosis. In these highly malignant cells,CD95 mediates migration/invasion. Binding of CD95 by the CD95L activatesPI3K and ILK, thereby leading to inhibition of GSK3β and to theinduction of metalloproteinases. Irradiation of apoptosis-resistantcells increased expression of CD95 and CD95L, which in turn increasedmetalloproteinase expression and subsequently, migration/invasion. In asyngenic mouse model of intracranial gliomas, CD95 and CD95L expressionwas upregulated upon interaction with the surrounding stroma.Neutralization of CD95 activity dramatically reduced the number of cellsinvading the contralateral hemisphere.

SUMMARY

The present invention provides a method for treating an individual withhigh grade glioma, comprising administering an agent that neutralizesCD95 activity to an individual with high grade glioma.

In one embodiment, the agent that neutralizes CD95 activity is acompound that prevents CD95 from binding to CD95L or disrupts aCD95/CD95L complex.

In one embodiment the compound binds to CD95 or binds to CD95L or bindsto the CD95/CD95L complex.

In another embodiment, the compound is an antibody that binds to CD95.In another embodiment, the compound is an antibody that binds to CD95L.The antibodies according to the invention may be monoclonal, polyclonalor chimeric. However, also single chain antibodies or functionalantibody fragments may be used. In one embodiment, the antibody thatbinds to CD95 is Nok1. Thus, the present invention contemplates anyneutralization agent or “inhibitor” of CD95 or CD95L or CD95R (the CD95receptor) activity.

For the production of antibodies according to the invention, varioushosts including goats, rabbits, rats, mice, humans, and others, may beimmunized by injection with the protein or any fragment or oligopeptidethereof which has immunogenic properties. Depending on the host species,various adjuvants may be used to increase immunological response. It ispreferred that the peptides, fragments or oligopeptides used to induceantibodies to the protein have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids.Monoclonal antibodies to the proteins may be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique. Inaddition, techniques developed for the production of ‘chimericantibodies’, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used. The antibodies may be used with orwithout modification, and may be labeled by joining them, eithercovalently or non-covalently, with, for example, a reporter molecule.

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the protein, ie. CD95 and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering protein epitopes are preferred, but acompetitive binding assay may also be employed.

One inhibitor may be a CD95-ligand (Fas ligand; APO1 ligand) inhibitor.For example, CD95-ligand inhibitors may be selected from (a) aninhibitory anti-CD95 ligand-antibody or a fragment thereof; (b) asoluble CD95 receptor molecule or a CD95 ligand-binding portion thereof;and (c) a Fas ligand inhibitor selected from FLINT, DcR3 or fragmentsthereof.

Also contemplated are inhibitory anti-CD95L-antibodies andantigen-binding fragments thereof and soluble CD95R molecules orCD95L-binding portions thereof. Examples of suitable inhibitoryanti-CD95L antibodies are disclosed in EP-A-0 842 948, WO 96/29350, andWO 95/13293 as well as chimeric or humanized antibodies obtainedtherefrom, cf. e.g. WO 98/10070, all of which are incorporated herein byreference. Further preferred are soluble CD95 receptor molecules, e.g. asoluble CD95 receptor molecule without transmembrane domain as describedin EP-A-0 595 659 and EP-A-0 965 637 or CD95R peptides as described inWO 99/65935, all of which are herein incorporated by reference.

In one embodiment, a CD95L inhibitor which comprises an extracellulardomain of the CD95R molecule, such as amino acids 1 to 172 (MLG . . .SRS) of the mature CD95 sequence according to U.S. Pat. No. 5,891,434,which is incorporated herein by reference, which may be fused to aheterologous polypeptide domain, particularly a Fc immunoglobulinmolecule including the hinge region e.g. from the human IgG1 molecule.One fusion protein comprises an extracellular CD95 domain and a human Fcdomain is described in WO 95/27735, which is herein incorporated byreference. Thus, according to a preferred embodiment, the agent whichbinds to CD95L is a fusion protein comprising an extracellular CD95domain and a human Fc domain. According to an especially preferredembodiment, the agent that binds to CD95L is APG101 (CD95-FC, SEQ IDNO:1). APG101 comprises the domains CD95R-ECD (amino acids 17-172; ECDextracellular domain) and IgG1-Fc (amino acids 173-400).

APG101 and derivatives thereof are disclosed in WO95/27735 andWO2004/085478 which herein incorporated by reference.

The Fas ligand inhibitor FLINT or DcR3 or a fragment, e.g. solublefragment thereof, for example the extracellular domain optionally fusedto a heterologous polypeptide, particularly a Fc immunoglobulin moleculeis described in WO 99/14330 or WO 99/50413 which are herein incorporatedby reference. FLINT and DcR3 are proteins which are capable of bindingthe CD95 ligand.

In a further embodiment of the present invention, the inhibitor is aCD95R inhibitor which may be selected from (a) an inhibitory anti-CD95receptor-antibody or a fragment thereof; and (b) an inhibitory CD95ligand fragment. A fragment of an inhibitory anti-CD95 receptor antibodyaccording to the in invention preferably presents the same epitopebinding site as the corresponding antibody does. In another embodimentof the invention a fragment of an inhibitory anti-CD95 receptor antibodyhas substantially the same CD95R inhibiting activity as thecorresponding antibody. An inhibitory CD95 ligand fragment preferablypresents substantially the same inhibiting activity as the correspondinginhibitory CD95 ligand fragment does.

Examples of suitable inhibitory anti-CD95R-antibodies and inhibitoryCD95L fragments are described in EP-A-0 842 948 and EP-A-0 862 919 whichare herein incorporated by reference.

In still a further embodiment of the present invention the inhibitor isa nucleic acid effector molecule. The nucleic acid effector molecule maybe DNA; RNA, PNA or an DNA-RNA-hybrid. It may be single stranded ordouble stranded. Expression vectors derived from retroviruses,adenovirus, herpes or vaccina viruses or from various bacterial plasmidsmay be used for delivery of nucleotide sequences to the targeted organ,tissue or cell population. Such constructs may be used to introduceuntranslatable sense or antisense sequences into a cell. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

The nucleic acid effector molecule may be in particular selected fromantisense molecules, RNAi molecules and ribozymes which are capable ofinhibiting the expression of the CD95R and/or CD95L gene. See, forinstance, U.S. 20060234968, which is incorporated herein by reference.In another embodiment, the high grade glioma is a WHO Grade III or IVglioma. In a preferred embodiment, the high grade glioma is a WHO GradeIV glioma.

According to an especially preferred embodiment of the invention theagent neutralizing CD95 activity prevents an interaction between CD95and the protein p85 of PI3K.

According to the present invention CD95 activity can be determined bythe person skilled in the by any kind of suitable assay, such asoutlined in Example 25 or Example 26.

The present invention also provides a pharmaceutical composition,comprising at least one agent that neutralizes CD95 activity to anindividual with high grade glioma. In one embodiment, the agent is Nok1or CD95-FC (APG101). CD95-FC binds to CD95L and thereby prevents itsbinding to CD95.

In another embodiment, the pharmaceutical composition comprises at leastone agent that binds CD95 and at least one other agent that binds toCD95L. The present invention also provides a method for treating apatient with glioma by administering one of the pharmaceuticalcompositions contemplated herein. In one embodiment, the glioma is aGrade III or Grade IV WHO-classified glioma, or a “high grade” glioma.

In another preferred embodiment the pharmaceutical composition maycomprise further active agents for the treatment of cancer and inparticular for the treatment of high grade glioma.

The pharmaceutical compositions may be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. The compositions may be administered to a patientalone or in combination with other agents, drugs or hormones. Thepharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations, which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of preadipocytecell lines or in animal models, usually mice, rabbits, dogs or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans. Atherapeutically effective dose refers to that amount of activeingredient, for example a nucleic acid or a protein of the invention oran antibody, which is sufficient for treating a specific condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratiobetween therapeutic and toxic effects is the therapeutic index, and itcan be expressed as the ratio, LD50/ED50. Pharmaceutical compositions,which exhibit large therapeutic indices, are preferred. The dataobtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage from employed,sensitivity of the patient, and the route of administration. The exactdosage will be determined by the practitioner, in light of factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors, which may be takeninto account, include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek or once every two weeks depending on half-life and clearance rateof the particular formulation. Normal dosage amounts may vary from 0.1to 100,000 micrograms, up to a total dose of about 1 g, depending uponthe route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and generallyavailable to practitioners in the art. Those skilled in the art employdifferent formulations for nucleotides than for proteins or theirinhibitors. Similarly, delivery of polynucleotides or polypeptides willbe specific to particular cells, conditions, locations, etc.

Another aspect of the present invention relates to a method forscreening for an agent, which modulates/effects, preferably neutralizesthe activity of CD95, comprising the steps

-   -   (a) incubating a mixture comprising        -   (i) CD95 or a functional fragment thereof        -   (ii) a candidate agent    -   under conditions whereby CD95 or a functional fragment thereof        has a reference activity;    -   (b) detecting the activity of CD95 or a functional fragment        thereof to determine an activity in the presence of the agent;    -   (c) determining a difference between the activity in the        presence of the agent and the reference activity.

According to a preferred embodiment of such an assay, an agent to bescreened for disrupts the interaction between CD95 and PI3K, preferablybetween CD95 and the p85 subunit of PI3K.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sensitivity to CD95-induced death and expression of CD95.

(a) The glioblastoma cell lines A172, T98G and LN18 were incubated withthe indicated doses of LZ-CD95L (ng/ml), Staurosporin (1 μM), or leftuntreated (Co). After 24 h and 48 h DNA fragmentation was analyzed byFACS. (b) FACS analysis of CD95 surface expression in the A172, T98G andLN18. (c) Spheroid cultures were embedded into a collagen matrix andtreated with LZ-CD95L (5 ng/ml). Invasion of single cells into thematrix was observed with a time-lapse microscope over 24 h. The distanceof invading cells (n=10 per spheroid; 3 spheroids per treatment) to theborder of the spheroid was depicted in the graph mean±S.E., *P<0.05. (d)Representative phase-contrast pictures of T98G and LN18 spheroids at 0 hand 24 h after treatment. Results are representative of threeindependent experiments.

FIG. 2: Cells resistant to CD95-induced death upregulate MMP-2 andMMP-9.

T98G (a) and LN18 2(b) cells were treated with α-Apo-1 (1 μg/ml) for theindicated times. Expression of MMP-2 and MMP9 was measured byquantitative real-time PCR. Data are results from five independentexperiments as mean+S.E., *P<0.05, **P<0.01.”

FIG. 3: CD95-induced migration is mediated via activation of ILK/AKT andthe inhibition of GSK3β

(a) GSK3β was phosphorylated and thereby inhibited in T98G and LN18cells upon treatment with α-Apo-1 (1 μg/ml). (b) T98G cells wereinfected with an empty lentiviral vector (Co) or a constitutively activeGSK3β mutant (GSK S9A). GSK S9A infected T98G cells migratedsignificantly less than their empty vector counterparts upon treatmentwith α-Apo-1 (2 μg/ml) or LZ-CD95L (CD95L, 5 ng/ml) at 36 h. Aneutralizing antibody to CD95L (Nok1 10 μg) blocked CD95L-inducedmigration of vector and GSK S9A infected cells. (c) GSK S9A infectedT98G cells and the respective controls were stimulated with theindicated doses of LZ-CD95L (ng/ml) for 48 h and DNA fragmentation wasanalyzed by FACS. (d) Growth curves of T98G Co and GSK S9A are shown.(e) The ILK inhibitor (KP-SD1, 10 μM) blocked α-Apo-1-(2 μg/ml) andLZ-CD95L-induced (5 ng/ml) migration of T98G cells. (f) The PI3Kinhibitor (LY 290059, 25 μM) blocked α-Apo-1-induced activation of AKTand inhibition of GSK3β in T98G cells. (g) Active β-catenin (green) wastranslocated to the nucleus upon stimulation with α-Apo-1 (1 μg/ml),phospho-GSK3β (red) did not change upon CD95 stimulation. DAPI (blue)was used to visualize the nuclei in T98G cells. (h) Cleavage ofCaspase-8 in T98G, LN18 and Jurkat 16 (J16) was detected upon CD95stimulation by Western Blotting. (i) The ERK inhibitor (PD 98059, 25 μM)did not interfere with the CD95-induced migration. Results are expressedas mean±S.E., *P<0.05, **P<0.01, and are representative of twoindependent experiments. P: phosphorylation, T: total.

FIG. 4: Expression of the CD95/CD95L system in primary glioblastomas

FACS analysis of CD95 surface expression in primary astrocytoma,oligondendroglioma and glioblastoma cell lines with low passages (≦4).For DNA fragmentation cells were treated with indicated doses ofLZ-CD95L (CD95L) for 24 h and 48 h and analyzed by FACS.

FIG. 5: Function of the CD95/CD95L system in apoptosis-resistant primaryglioblastomas

(a) FACS analysis of CD95 surface expression in primary NCH (89, 125 and270) glioblastoma cell lines highly passaged (≧50). For DNAfragmentation cells were treated with indicated doses of LZ-CD95L(ng/ml) for 24 h and 48 h and analyzed by FACS Results are depicted asmean±S.D. and are representative for three independent experiments. (b)NCH 89, 125 and 270 cells showed an upregulation of MMP-2 and MMP9 mRNAlevels 40 h after α-Apo-1 (1 μg/ml) treatment as detected byquantitative real-time PCR. Results are depicted as mean±S.E., *P<0.05,**P<0.01, ***P<0.005 and are representative for three independentexperiments. (c) Migration assay for NCH 89, 125 and 270 cells treatedwith α-Apo-1 (0.1 μg/ml+Protein A). Results are expressed as mean±S.E.,*P<0.05, **P<0.01 and are representative of two independent experiments.

FIG. 6: γ-irradiation-induced migration in T98G and primary glioblastomacells

(a+b) T98G cells were γ-irradiated at the indicated doses andmRNA-levels of CD95 and CD95L (a) and MMP-2 and -9 (b) were determinedby quantitative real-time PCR after 40 h. Results are depicted asmean±S.E., *P<0.05, **P<0.01 and are representative of three independentexperiments. (c+d) Induction of migration in T98G (c) and primary NCHcells (NCH 125, 270 and 89) (d) upon γ-irradiation. A neutralizingantibody to CD95L (Nok1, 10 μg) blocked γ-irradiation-induced migration.Results are expressed as mean±S.E., *P<0.05 and representative for twoindependent experiments. (e) CD95L scoring of 9 different recurrentgliomas following radiotherapy. 3 areas per tumor were analyzed and theCD95L positive cells were counted and scores assigned according to thenumber of positive cells. Recurrent tumors analysed were NCH 907, 202,910, 860, 655, 408, 30, 388 and 715. (f) Stainings were performed onconsecutive recurrent tumor tissue sections for CD95L, CD95 and MMP9, aswell as the double staining for GFAP (green) and CD95L (red).

FIG. 7: CD95 and CD95L are upregulated on murine glioma cells in vivoand induce migration

(a) Immunohistochemical staining for CD95L of primary GBM. (b-c) CD95and CD95L surface expression on the murine glioma cell line SMA-560 wasdetermined under normal cell culture conditions, after the formation ofspheroids or following intracranial implantation. Changes of CD95 (b)and CD95L (c) (GeoMean from BD CellQuest Pro) under the aforementionedconditions were normalized to expression levels under cell cultureconditions. Results represent three independent experiments and areexpressed as mean±S.D., *P<0.05. (d) Spheroid cultures were embeddedinto a collagen matrix and treated with antibodies to CD95 (Jo2), aneutralizing antibody to CD95L (MFL3) or the appropriate isotype controlantibody at the indicated concentrations. The migration of cells wasmonitored over 48 h and the distance of cells to the spheroid's borderis depicted (n=10 cells, 3 spheroids per treatment). (e) Experimentalsetup. Migration of tumor explants into collagen after treatment witheither MFL3 or the appropriate isotype control is depicted as describedabove (n=10, 3 spheroids per treatment). (f) Numbers of SMA-560 cells inthe contralateral hemisphere of Vm/Dk mice either treated with MFL3 orthe isotype control antibody were counted and normalized to tumorsurface. Shown results are representative of two independent experimentsand expressed as mean±S.D., *P<0.05, **P<0.001.

FIG. 8: Inhibition of GSK3β is caspase independent

(a) GSK3β is phosphorylated and thereby inhibited in T98G and LN18 cellsupon treatment with LZ-CD95L (5 ng/ml). (b) Preincubation with thecaspase inhibitor zVAD-fmk (40 μM) did not interfere with α-Apo-1 (2μg/ml)-induced inhibition of GSK3β. (c) PED knockdown with siRNA blockedthe activation of ERK, but did not change the α-Apo-1-induced inhibitionof GSK3β. Results are representative of two independent experiments.

FIG. 9: Expression and function of the CD95/CD95L system in primaryglioblastomas

(a-b) FACS analysis of CD95 surface expression in primary glioblastomacell lines (NCH 82, 37, 125, 149, 89, 156, 270 and 199) with highpassages (≧50). For DNA fragmentation cells were treated with indicateddoses of LZ-CD95L (ng/ml) for 24 h and 48 h and analyzed by FACS.Results are given as mean±S.D. and are representative of two independentexperiments. (c) mRNA levels of CD95 and CD95L in human tumor tissue,determined by quantitative real-time PCR. Results are representative oftwo independent experiments.

FIG. 10: Influence of irradiation on primary glioblastoma migration

FACS analysis of CD95 surface expression in primary glioblastoma celllines (NCH 342, 354, 357, 378, 417, 419 and 2421). Induction ofmigration in these primary NCH cells upon γ-irradiation, which could beblocked by a neutralizing antibody to CD95L (Nok1, 10 μg). Results areexpressed as mean±S.E., *P<0.05, **P<0.01, ***P<0.005 and representativeof two independent experiments.

FIG. 11: CD95 triggers invasion of apoptosis resistant cells via MMPs

(A) The glioblastoma cell lines T98G and LN18 were incubated with theindicated concentrations of CD95L-T4, Staurosporin (St., 1 μM) or leftuntreated (Co). After 24 h DNA fragmentation was analyzed by FACS (upperpanel). FACS analysis of CD95 surface expression in the T98G and LN18(lower panel). (B) T98G and LN18 cells were treated with CD95L-T4 orleft untreated, to detect single cell migration through a Boyden chamberwith 8 μm pore size. (C) T98G cells were treated with CD95L-T4 for 24 hor left untreated. Thereafter, MMP-9 activity was assessed by GelZymography. (D and E) T98G and LN18 cells were treated with αApo-1 for48 h or left untreated. Expression of MMP-2 and MMP-9 was measured byquantitative real-time PCR. Data are results from five independentexperiments as mean±S.E., *P<0.05. (F) T98G cells were transfected witha siRNA pool against MMP-2 and MMP-9 (shMMPs) or with Lipofectaminealone (Lipo). 48 h after transfection, cells were treated with CD95L-T4,48 h afterwards migration was measured in a two dimensional migrationassay. FIG. 11 (G) Expression of MMP-2 and MMP-9 as measured byquantitative-RT-PCR. Results are expressed as mean±S.E., *P<0.05;**P<0.001; ***P<0.0001 and are representative of at least twoindependent experiments.

FIG. 12: CD95 triggers invasion of primary apoptosis-resistant gliomacells via MMPs

(A) The short term cultured cell lines NCH89, 125 and 270 were incubatedwith the indicated concentrations of CD95L-T4, Staurosporin (St., 1 μM)or left untreated (Co). After 24 h DNA fragmentation was analyzed byFACS (upper panels). FACS analysis of CD95 surface expression in theNCH89, 125 and 270 (lower panels). (B) NCH cells were treated for 48 hwith αApo-1 or left untreated, to detect single cell migration through aBoyden chamber with 8 μm pore size. Results are expressed as mean±S.E.,**P<0.01 and are representative of three independent experiments. (C andD) NCH89, 125 and 270 cells were treated with a Apo-1 for 48 h.Expression of MMP-2 (C) and MMP-9 (D) was measured by quantitativereal-time PCR. (E) NCH125 cells were transfected with a siRNA poolagainst MMP-2 and MMP-9 (shMMPs) or with Lipofectamine alone (Lipo). 48h after transfection, cells were treated with CD95L-T4, migration wasmeasured in a two dimensional migration assay 48 h later. (F) Expressionof MMP-2 and MMP-9 as measured by quantitative-RT-PCR. Results areexpressed as mean±S.E., *P<0.05; **P<0.001; ***P<0.0001 and arerepresentative of at least two independent experiments.

FIG. 13: CD95 induces migration via the PI3K/AKT/GSK3β Pathway

(A) Phosphorylation of AKT and ERK is shown in T98G and LN18 cells upontreatment with CD95L-T4 at the indicated time points. (B) In T98G, LN18and NCH125 and 270 cells, but not in NCH89, phosphorylation of AKTexhibited a concentration-dependent bell-shape after stimulation withCD95L-T4, respectively. (C) In T98G cells treatment with CD95L-T4 (10ng/ml) and αApo-1 (1 μg/ml) induced GSK3b phosphorylation. Kinetics ofGSK3b-inhibition exhibited a bell-shaped curve. (D) T98G cells wereinfected with an empty lentiviral vector (Co) or a constitutively activeGSK3b mutant (GSK S9A). At 36 h GSK S9A infected T98G cells migratedsignificantly less than their empty vector counterparts upon treatmentwith αApo-1 or CD95L. A neutralizing antibody to CD95L (Nok1) blockedCD95L-induced migration of vector- and GSK S9A-infected cells. (E)b-Catenin accumulated in the cytosol of T98G cells 30 min aftertreatment with CD95L-T4 (10 ng/ml). (F) Active b-catenin (green)translocated into the nucleus upon stimulation with αApo-1. DAPI (blue)and P-GSK3β (red) was used to visualize the nuclei and the cytosol,respectively in T98G cells. (G and H) 24 h after transient transfectionof T98G with the b-Catenin/TCF transcriptional reporter (TOP-FLASH; (G),the control plasmid (FOP-FLASH; G), or the NFkB-reporter construct(NFkB-Luc; H) cells were treated with CD95L-T4 (G and H), LiCl (G) orleft untreated (G and H). Luciferase activity was assayed 12 h (G) or 8h (H) afterwards and normalized to renilla luciferase expression.Results are expressed as mean±S.E., *P<0.05; **P<0.001; ***P<0.0001 andare representative of two independent experiments. P: phosphorylated, T:total.

FIG. 14: Src and p85 associate to CD95 to activate AKT

(A and B) LN18 and T98G cells were stimulated with CD95L-T4 for theindicated time points and concentrations. On the left panels CD95 wasimmunoprecipitated and the immunoprecipitates immunoblotted withanti-p85, anti-CD95, and anti-Src antibodies. On the middle panels, p85was immunoprecipitated and the immunoprecipitates probed with anti-p85and anti-CD95 antibodies. Protein-A beads are included as a negativecontrol. Whole cellular lysates (WCL) probed with anti-p85, anti-CD95and anti-Src are shown on the right panels. (C) T98G cells were treatedfor the indicated times and concentrations with CD95L-T4. CD95 wasimmunoprecipitated and immunoprecipitates were immunoblotted withanti-Yes antibody. (D) T98G and NCH125 cells were transfected with siRNAagainst Yes or Fyn or with Lipofectamine alone (Lipo). 72 h aftertransfection, cells were treated with CD95L-T4, 24 h afterwardsmigration was measured in a two dimensional migration assay. (E)Expression of Yes-mRNA as measured by quantitative-RT-PCR and Yesprotein levels as assessed by FACS analysis is shown. (F) T98G andNCH125 cells were transfected with siRNA against Yes, a Yesoverexpression plasmid (pCMV-Yes) or both. 72 h after transfection,cells were treated with CD95L-T4, 24 h afterwards migration was measuredin a two dimensional migration assay. (G) Yes siRNA and the PI3Kinhibitor (LY 290059) blocked CD95-induced phosphorylation of AKT. P:phosphorylated; T: total; *: specific band; n.s.: unspecific band.

FIG. 15: Inefficient DISC formation in apoptosis resistant glioma cells

(A) 48 h after transient transfection of T98G and NCH125 with either aHA-PTEN (PTEN) or the empty vector (Mock), cells were treated withCD95L-T4 (500 ng/ml). To detect cell death in PTEN-overexpressing cells,an intracellular FACS staining against the HA-tag was performed.Thereafter forward side scatter analysis was performed in HA-positivecells. Results are expressed as mean±S.E. and are representative of twoindependent experiments. (B) T98G cells were treated for 1 h withαApo-1, zVAD-fmk, a combination of zVAD-fmk and αApo-1 or left untreated(Co). Phosphorylation of GSK3β was analyzed by Western Blot. (C)Cleavage of caspase-8 in T98G, LN18 and Jurkat 16 (J16) was detected byWestern Blot analysis at 5 min after CD95 stimulation. (D) In T98G andLN18 cells treated with CD95LT4 for 5 min, either CD95 (upper panels) orcaspase-8 (lower panels) were immunoprecipitated. The immunoprecipitateswere immunoblotted with anti-FADD antibody and anti-CD95 (upper panels)and with anti-CD95 and anti-caspase-8 (lower panels). Jurkat cells wereincluded as a positive control. (E) T98G cells were transfected witheither Yes shRNA or a non-targeting shRNA as a negative control. After72 h, cells were treated with CD95L-T4 or left untreated andimmunoprecipitation of CD95 was performed, the immunoprecipitates werethen immunoblotted with anti-FADD antibody, the IgG heavy chain servesas loading control. On the right, knockdown efficiency assessed byquantitative-RTPCR is shown. (F) Quantitative expression of FADD, Yesand Fyn in T98G and LN18 cells was measured by FACS analysis. Resultsare expressed as mean±S.D., *P<0.05 and are representative of threeindependent experiments. MFI: mean fluorescence intensity P:phosphorylated; T: total

FIG. 16: Schematic model for CD95 signalling of invasion in glioblastoma

CD95L induces recruitment of the Src family member Yes and the p85subunit of PI3K (depicted here by its two subunits: p85 and p110) toCD95, thereby activating AKT. Activated AKT phosphorylates andinactivates GSK3b, allowing b-catenin translocation into the nucleus,where it induces transcription of MMPs. This signalling pathway could beblocked by siRNA against Yes, or MMP-2 and -9, the PI3K-specificinhibitor (Ly290059) or by lentiviral infection with a dominant activemutant of GSK3β (S9A).

FIG. 17: Expression of CD95L, Yes and Phospho-Src in clinical samples ofGBM

(A) Representative immunohistochemical staining of CD95L (red) inprimary human GBM. Note the increased expression of CD95L at thetumor/host interface (A.b) compared to more solid tumor areas (A.a*) orbrain parenchyma (A.c). (B) Immunohistochemical staining ofphosphorylated Src family kinases (p-Src; B.a,c,e,f) and Yes(B.b,d,g,h). B.a-d, Overview of tumor infiltration zone, note robustphosphorylation of Src and expression of Yes in zone of tumor/hostinteraction (to the left) and reduced or no p-Src in solid tumor areas(to the right*). Yes expression was found at the tumor/host interface(B.b, d and g) and in scattered solid tumor areas (B.h). Strongphosphorylation of Src in tumor cells in solid tumor areas (B.f) andinfiltration zone (B.e).

FIG. 18: CD95 and CD95L are upregulated on murine glioma cells in vivoand induce migration

(A and B) CD95 and CD95L surface expression on the murine glioma cellline SMA-560 was determined under normal cell culture conditions, afterthe formation of spheroids or following intracranial implantation.Changes were normalized to cell culture conditions levels. Resultsrepresent three independent experiments and are expressed as mean±S.D.,*P<0.05. (C) Spheroid cultures were embedded into a collagen matrix andtreated with antibodies to CD95 (Jo2), a neutralizing antibody to CD95L(MFL3) or the appropriate isotype control antibody at the indicatedconcentrations. The migration of cells was monitored over 48 h and thedistance of cells to the spheroid's border is depicted (n=10 cells, 3spheroids per treatment). (D) Experimental scheme. Migration of tumorexplants into collagen after treatment with either MFL3 or theappropriate isotype control is depicted as described above (n=10, 3spheroids per treatment). (E) Representative pictures ofGFP-immunostained Isotype- or MFL3-treated SMA-tumors. The tumor—bearing(ipsilateral) and contralateral hemisphere are shown. Numbers of SMA-560cells (GFP-positive) in the contralateral hemisphere of Vm/Dk miceeither treated with MFL3 or the isotype control antibody were countedand normalized to tumor area. Shown results are representative of twoindependent experiments and expressed as mean±S.D., *P<0.05, **P<0.001.Scale bar: 100 μm

FIG. 19: Generation of CD95L-T4

Localization of the N- and C-terminal amino acids within the TRAIL-RBD,forming an antiparallel β. (A) Enlargement of the TRAIL-RBD-structure(N-ter-minus: green; C-terminus: red). (B) Position of the N- andC-terminus within the structure of the TRAIL-RBD. Upper row: side viewof the central axis of the TRAIL-RBD. Lower row: top view of the centralaxis of the TRAIL-RBD. Left to right: mono-, di- and trimer. (C)CD95L-T4 amino acid sequence (SEQ ID NO:26). The signal peptide isunderlined (Met1-Gly20). The N-terminal (green arrow) and C-terminalamino acids (red arrow) of the CD95L-RBD are proposed to form anantiparallel .beta.-strand. The T4-Foldon sequence is printed in blue(italics). (D) Model of TRAIL-T4-DR5 complex. Upper Row: side view ofTRAIL-DR5 co-complex with the T4-Foldon (light blue) positioned abovethe TRAIL-RBD (dark blue). The DR5 chains are colored in dark red. TheN-terminal amino acids of the TRAIL-RBD and the T4-Foldon are colored ingreen; the C-terminal amino acids are colored in red. Upper row: sideview of the central axis of the TRAIL-RBD. Lower row: top view of thecentral axis of the TRAIL-RBD. Left: ribbon model. Right: surface model.(E) Affinity purification of CD95L-T4. CD95L-T4 containing supernatantfrom transiently transfected Hek293T cells was affinity purified usingStreptactin Sepharose. Proteins specifically eluted by desthiobiotinwere subsequently analyzed by SDS-PAGE and silver staining. The positionof CD95L-T4 is indicated by an arrow. CD95L-T4 shows an apparentmolecular weight of approximately 30 kDa. The difference towards thetheoretical molecular weight of 23 kDa is probably due to glycosylation.(F) Analysis of purified CD95L-T4 by size exclusion chromatography(SEC). For determination of the native apparent molecular weightCD95L-T4 was separated by SEC using a Superdex 200 column. The graphshows the elution profile (OD 280 nm) of Streptactin-purified CD95L-T4.Fraction B3-C2 collected from SEC (see B) was analyzed by SDS-PAGE andsilver staining. (G) Determination of the apparent molecular weight.Calibration curve of the Superdex 200 column based on retention volumesof the indicated proteins. The apparent native molecular weight ofCD95L-T4 is 90.3 kDa, indicating a stable trimeric protein. AP-G101antagonizes the apoptosis inducing activity of CD95L-T4 in Jurkat cells.(H) CD95L-T4 induces apoptosis in Jurkat cells in a dose dependentmanner, as shown by an increase in Caspase 3/7 activity. Addition of 10μg/ml anti-StrepMAB, an anti-body crosslinking the CD95L-T4 trimers,augments the extent of apoptosis. (I) The apoptosis inducing activity of250 ng/ml CD95L-T4 in Jurkat cells is antagonized by APG101 in a dosedependent manner.

FIG. 20: CD95/CD95L system and sensitivity to apoptosis in primaryglioblastomas

(A and B) FACS analysis of CD95 surface expression in primaryglioblastoma cell lines (NCH82, 37, 125, 149, 89, 156, 270 and 199) withhigh passages (≧50). For DNA fragmentation cells were treated withindicated doses of CD95L (ng/ml) for 24 h and 48 h and analyzed by FACS.Results are given as mean±S.D. and are representative of two independentexperiments. (C) mRNA levels of CD95 and CD95L in human tumor tissue,determined by quantitative real-time PCR. Results are representative oftwo independent experiments.

FIG. 21: Modulators of CD95-mediated PI3K activation

(A) LN18 cells were incubated with the indicated concentrations ofCD95L-T4 with or without a neutralizing antibody to CD95L (Nok1),αAPO-1, Staurosporin (St., 1 μM), or left untreated (Co). After 24 h and48 h, DNA fragmentation was analyzed by FACS. (B) T98G and the shortterm cultured cell lines, NCH89 and 125 were incubated with theindicated concentrations of αApo-1, Staurosporin (St., 1 μM), or leftuntreated (Co). After 24 h and 48 h, DNA fragmentation was analyzed byFACS. (C) NCH125 and NCH270 cells were treated for 48 h with CD95L-T4 orleft untreated, to detect single cell migration through a Boyden chamberwith 8 μm pore size. (D) GSK phosphorylation in T98G cells measured byintracellular FACS staining. (E) GSK S9A-infected T98G cells and therespective controls were stimulated with the indicated doses of CD95L(ng/ml) for 48 h and DNA fragmentation was analyzed by FACS. Growthcurves of T98G Co and GSK S9A are shown. (F) T98G and NCH125 cells weretransfected with siRNA against Yes or Fyn or with Lipofectamine alone(Lipo). Expression of Fyn as measured by quantitative-RT-PCR is shown.Results are expressed as mean±S.E., *P<0.05; **P<0.001; ***P<0.0001.

FIG. 22: Table 1

Summary of the clinical data from tumor patients.

DETAILED DESCRIPTION

It is generally appreciated that the CD95/CD95L complex inducesapoptosis¹⁸. However, there is growing evidence that CD95 can mediateapoptosis-independent processes such as proliferation, angiogenesis,fibrosis, and inflammation^(19,20). Over-expression of CD95 in Lewislung carcinoma cells resulted in a survival advantage of tumor cells invivo²¹. Along the same lines, triggering of CD95 has been reported todrive cell cycle progression in glioma cells²². In malignantastrocytoma, CD95 ligation promoted expression of pro-inflammatorychemokines and angiogenesis²³⁻²⁵. Here we report that triggering of CD95in glioblastomas initiates a cascade of signaling events ultimatelyleading to increased invasiveness. CD95-induced migration was firstobserved in cultured renal tubular cells²⁶ and has recently beenreported for ovary, breast, lung and kidney tumor cells¹³. In the latterstudy, a serum-gradient was added to the CD95 stimulus in order toinstigate and direct cell migration¹³. Glioblastoma cells, on thecontrary, are characterized by their invasion-prone phenotype and domigrate in the absence of an additional stimulus.

Thus, the present invention concerns methods for blocking in vitro,ex-vivo and in vivo the highly invasive glioblastoma behaviour by thesole neutralization of CD95 activity. The present invention contemplatesany method or mechanism for neutralizing or otherwise blocking CD95activity, such as by using an antibody that binds to it or bydownregulating or inhibiting CD95 gene expression or CD95 mRNAtranscript translation.

To invade and spread into the surrounding normal brain, tumor cells needto digest components of the extracellular matrix, including fibronectin,laminin, and type IV collagen. The best characterized family ofECM-degrading enzymes is the MMP family. MMP9 deficient glioblastomasare less invasive in vitro and in vivo²⁷. Glioblastomas producesignificantly higher levels of MMP9 than do lower-grade gliomas andnormal brain tissue²⁷. Levels of MMP9 increased during the growth ofglioblastoma cells that had been implanted intracranially in nudemice²⁸. These proteases have also a role in establishing and maintaininga microenvironment that facilitates tumor-cell survival. Accordingly,MMPs regulate tumor angiogenesis and inhibition of MMP9 reducedcapillary-like structures in mixed-cultures of endothelial and gliomacells²⁷. The same features apply to CD95L expression: (i) levelspositively correlate with the degree of malignancy^(23,29,30); (ii)levels increase after intracranial inoculation; (iii) in human specimensof GBM one of its preferential localization is at the tumor vessels.

Here the present inventions demonstrates that triggering of CD95increases mRNA expression of MMP9 and MMP2 in established glioblastomacell lines and primary cultures and knockdown of MMP2 and MMP2 blocksCD95-induced migration.

The promoter region of MMP9 contains putative binding sites for AP-1,NFκB, Sp1 and AP-2³¹. The AP-1 transcription complex plays an essentialrole in stimulating transcription of MMP9^(31,32). AP-1 driventranscription of MMP9 in glioblastoma cells has been previously reportedto be downstream of the PI3K/ILK/GSK pathway^(9,10) or instead mayrequire ERK and JNK activity³³.

c-Jun, a putative component of the AP-1 transcription complex, has beenidentified as one of the most highly induced TCF/β-catenin targetgenes^(36,37). Inhibition of GSK3β, as reported here, allowsunphosphorylated β-catenin to accumulate and translocate into thenucleus, where it functions as a cofactor for transcription factors ofthe TCF/Lef family³⁵. In addition, activity of NFκB was concomitantlyobserved. Since CD95L-T4 decreases ERK activity, we believe that in T98Gcells AKT regulates NFκB activity through phosphorylation/activation ofthe IKK kinase, which in turn phosphorylates IκB and allows the releaseof activated NFκB⁶⁶. Alternatively, IκB can transactivate the p65subunit⁶⁴. In contrast, the induction of motility and invasivenesspreviously reported for tumor cell lines of endodermal or mesodermalorigin involves ERK, NFκB and Caspase-8 activity¹³.

In contrast, treatment of glioblastoma cells with CD95L did neithertrigger ERK activation nor caspase-8 cleavage. CD95-mediated invasion ofglioblastoma cells could not be blocked by an ERK or a general caspaseinhibitor. Instead, in these cells from neuroectodermal origininvasiveness was regulated via the PI3K/ILK/GSK pathway, as it could beblocked by a PI3K-, an ILK-inhibitor and by a dominant-active form ofGSK3β. PI3K activity is also required for association of the epidermalgrowth factor receptor (EGFR) which then increases expression of MMP9via ERK³⁴. Thus, CD95-mediated activity of PI3K facilitates anadditional increase in MMP9 activity by EGFR. Thus, activation of CD95induces migration/invasion through the PI3K/AKT/GSK3β/β-catenin/MMPand/or the PI3K/AKT/NFκB/MMP pathway.

GSK3β is found in a multiprotein complex with the adenomatous polyposiscolon (APC) tumor suppressor protein, axin and β-catenin. Inunstimulated cells, GSK3β phosphorylates β-catenin, marking it forubiquitination and subsequent degradation³⁵. Inhibition of GSK3βdestabilizes this degradation complex, allowing unphosphorylatedβ-catenin to accumulate and translocate to the nucleus where itfunctions as a cofactor for transcription factors of the T-cellfactor/lymphoid enhancing factor (TCF/LEF) family³⁵. C-Jun, a putativecomponent of the AP-1 transcription complex, has been identified as oneof the most highly induced TCF/β-catenin target genes^(36,37).

Phosphorylated c-Jun has recently been found to interact with TCF4 and,thereby, regulate intestinal tumorigenesis by integrating the JNK andGSK pathways. We hypothesize that high basal ERK and JNK activitiestogether with GSK3β inhibition determine the tumorigenic activity ofCD95. In this respect, the present inventors found that high basallevels of phosphorylated GSK3β positively correlate with the ability ofCD95 to increase migration (data not shown) while the levels ofCD95-surface expression did not have any influence. Accordingly,malignant gliomas exhibit greater free pools of unphosphorylatedβ-catenin than less malignant ones (own unpublished data).

In the past, several reports have pointed out an important role fortyrosine phosphorylation in CD95-induced signalling^(56,59). Thesepreliminary reports, however, suggested that CD95-induced tyrosinephosphorylation is a prerequisite for CD95-mediatedapoptosis^(56,60,69). Along this line, the phosphatases SHP-1, SHP-2 andSHIP were found to associate with CD95 to counteract survivalfactors-initiated pathways⁵⁴. Just recently, Src-induced tyrosinephosphorylation of caspase-8 was found to impair CD95-inducedapoptosis⁵³. We now describe a novel association of the Src familymember Yes and p85 with CD95. TRANCE, another TNF family member,activates PI3K through a signalling complex involving c-Src and TRAF6⁷¹.Inhibition of Fyn, another Src family member, decreased CD95-inducedmigration of glioblastoma cells, although not significantly. This can beexplained by the fact that Fyn is involved in EGFR-mediated signallingin neural cells and EGFR is a very important receptor for gliomainvasion that has been found in association with CD95. Thus inhibitionof CD95-mediated signalling might affect EGFR mediated signalling andvice versa. Whether another adaptor molecule is still missing in CD95'sPI3K-activation complex (PAC) remains subject of future studies.Alternatively, Yes and p85 might directly interact with CD95 through thepreviously identified phosphotyrosine containing motif in the DD ofCD95⁵⁴. Accordingly, in T98G cells, knockdown of Yes enabledCD95L-T4-induced recruitment of FADD to CD95 indicating that Yes andFADD might compete for binding to CD95. Along this line, analysis of Yesexpression levels revealed a much higher expression in T98G than in LN18cells. Most importantly, expression of Yes and phosphorylation of Srcfamily kinases was consistently found at the site of tumor/hostinteraction in clinical samples of GBM, indicating its involvement intumor invasion.

Barnhart et al. (2004)¹³ previously showed that exogenous CD95L inducesmigration of tumor cells from endodermal origin in vitro. In these cellsCD95L induces migration via caspase-8 and ERK¹³. These authors speculatethat CD95L might be involved in the tumor's escape to chemo- andradiotherapy, since both treatments increase expression of CD95L. In ourstudy we found that CD95L also induces migration of GBM cells. Beyondthis, the present study represents a significant conceptual advance inthe field of tumor biology since it shows for the first time that: (i)the sole interaction of tumor cells with the surrounding parenchymainduces expression of CD95L in tumor and host cells; (ii) in GBM cellsCD95L signals invasion via Yes/PI3K/MMPs and not via caspase-8/ERK as itis the case in tumor cells of endodermal origin; (iii) neutralization ofCD95 activity blocks the basal migration of GBM cells in vivo in a mousesyngenic model of GBM that mimics the clinical situation. In addition,this study shows that the molecular stoichiometry of the PI3K signallingcomponents seems to determine the cellular response to CD95.

In summary, the present data indicate that WHO Grade IV tumors areresistant to CD95-induced apoptosis but increased their invasioncapacity upon stimulation of CD95.

Despite the known clinical resistance to irradiation of GBM, the currenttherapy for GBM encompasses surgery followed by irradiation and adjuvantchemotherapy. The standard irradiation regimen uses an optimal dose of60 Gy usually given in daily fractions of 1.8 to 2 Gy for approximately6 weeks with concomitant focally directed radiotherapy. The target areais the enhanced area typically seen on MRI with an additional 2 to 3 cmmargin³⁸. This regimen has been developed based on the knowledge thatthe principal treatment failure in malignant gliomas is tumor recurrencewithin 2 cm of the original tumor site, occurring in approximately 80%to 90% of cases³⁸. Radiation induces damage by direct interaction withthe cellular target or indirectly through interaction with other atomsor molecules (e.g., water) within the cell to produce free radicals thatsecondarily affect critical structures. In addition, irradiation hasbeen shown to increase expression of death receptors and death ligands,which in some cases kill the cell via apoptosis³. The present inventionconfirms that irradiation of glioblastoma cells greatly increases thelevels of CD95 and CD95L. Nevertheless, cells remain resistant toradiation-induced damage. Instead, we show that irradiated cells exhibita higher CD95L dependent migration potential. Even cells that did notincreasingly migrate after the sole stimulation of CD95 did so afterirradiation. Thus, an additional irradiation-mediated increase of CD95levels or possible changes in the overall kinase activity might rendercells sensitive to CD95-induced migration. Along this line, therapeuticX-irradiation is the only environmental factor unequivocally linked to ahigher risk of brain tumors, including glioma, often within ten yearsafter therapy³⁹. Most importantly, in contrast to the original tumorswhere CD95 and CD95L were barely expressed within the tumor, inrecurrent GBM expression of CD95 and CD95L dramatically increased. Inline with the in vitro data, we did not detect apoptotic cells nearCD95L-positive cells, but instead an increased expression of MMP9.

Current experimental strategies to block glioblastoma invasion focus oninhibition of MMP activity by expression of the natural inhibitors TIMP2and TIMP4 or rely on direct gene targeting of MMP mRNA by antisensestrategies. However, while TIMP2 decreases angiogenesis and invasion italso protects tumor cells from apoptosis⁴⁰. Other strategies to inhibitMMP production employs targeting the signal-transduction pathwaysleading to their expression which are similarly not only involved in theinduction of tumor invasion but also in some basic neural functions,thus, making these strategies less attractive for clinical application.

By contrast, while CD95 activity is required for neurite remodelingduring embryonic brain development⁴¹ and for the clearance of damagedbrain cells in the diseased brain⁴²⁻⁴⁵ no CD95 activity is detectable inthe adult healthy brain. brain. Thus, targeting CD95 should have fewerside effects than other migration-inducing factors that are normallyinvolved in normal brain function.

Thus, CD95 appears as a very potent and attractive target for thefront-line therapy of human glioblastoma.

EXAMPLES Example 1 Reagents and General Procedures

Following antibodies were used: anti human-CD95L G247-4 (1:200), theneutralizing antibody to CD95L (Nok1), the anti murine-CD95 (Jo2), theanti murine-CD95L (MFL3) and the appropriate isotype control, a hamsterIgG1, λ1, were purchased from Becton Dickinson. Antibodies against CD95(α-Apo-1) was generated as described previously⁴⁶, phosphorylated GSK3β(P-Ser9-GSK3β, 1:1000), phosphorylated AKT (P-Ser473-KT, 1:1000), totalAKT (T-AKT, 1:1000), Src family kinases (Src, 1:1000), phosphorylatedSrc (P-Tyr416 1:50) and total β-Catenin (1:1000) were purchased from NewEngland Biolabs. Antibodies against CD95 (CD95, 1:1000), total GSK3β(T-GSK3β, 1:1000), phosphorylated ERK1/2 (P-ERK, 1:1000), total Yes(Yes, 1:1000 or 1:200), total Fyn (1:200) and total ERK (T-ERK, 1:1000)were purchased from Santa Cruz. Anti-GFAP (1:200) was purchased fromChemicon, anti-PI3K (p85 N-SH2 domain, 1:1000), anti-FADD (FADD, 1:1000)and active-β-Catenin (P-Ser37 or P-Thr41; 1:800) were purchased fromUpstate, anti-MMP9 GE-213 (1:100) was purchased from Oncogene, anti-GFP(1:400) was purchased from Molecular Probes, anti-GADPH was purchasedfrom Abcam and the anti-Caspase-8 antibody (1:10) was generated in ourlaboratory from hybridoma supernatant⁴⁷. The anti-PED antibody (1:2000)was kindly provided by Dr. Nerve Chneiweiss. For visualizing specificantibodies on histological stainings streptavidine-alkalinePhosphatase/FastRed both purchased from Dako were used. Forimmunofluorescence studies the monoclonal anti odies Alexa Fluor 488(1:500; Molecular Probes) and anti-Rhodamine (1:200; Dianova) were used.

The MAPK inhibitor, PD98059 (25 μM), the PI3K inhibitor Ly 290059 (25μM) and the pancaspase inhibitor zVAD-fmk (40 μM) were purchased fromCalbiochem. The ILK inhibitor (20 μM) was kindly provided from S.Dedhar. Cells were preincubated with inhibitors 30 minutes (LY 290059and zVAD-fmk) prior to treatment with either CD95L-T4 or αApo-1.Generation of Leucinezipper-CD95L (LZCD95L) was performed as previouslydescribed⁴¹. The blocking agents (zVAD-fmk, PD98059, LY 290059 andKP-SD1) were given 30 minutes before treatment with LZ-CD95L or αApo-1.Cells were lysed for further biochemical analysis. Protein extractionand immunoblotting was performed as previously described⁴¹.

Example 2 Primary Samples

Tissue specimens of NCH tumors were obtained intraoperatively afterinformed consent of the patients and approval of the local ethicscommittee. Fresh tissue was divided into two parts, one part toestablish primary tumor cultures and the other for RNA extraction.Clinical data of the respective patients concerning tumorclassification, age at surgery and sex are summarized in Table I (FIG.22).

Example 3 Animal Experiments

Animal experiments were approved by the German Cancer Research Centerinstitutional animal care and use committee and the RegierungspräsidiumKarlsruhe. For intracranial injections, 8- to 12-week-old inbred Vm/Dkmice were used. 5.000 SMA-560 cells were harvested by trypsinization,resuspended in 1 μl Dulbeccos modified eagle medium (DMEM supplementedwith 10% fetal bovine serum (FCS), 1% Penicillin/Streptomycin (PS) and1% L-Glutamine 200 mM) and loaded into a 10 μl Flexilfil syringe (WPI,Berlin, Germany). A burr hole was drilled 2.75 mm lateral to the bregmaand the needle was introduced to a depth of 3 mm. Mice were sacrificed7, 14 or 18 days after injections.

Example 4 Tumor Explants

Fourteen days after tumor inoculation, mice sacrificed and the tumorsextracted. Tumor explants were then incubated for 1 hour in eithermedium, medium plus isotype control antibody (10 μg/ml) or medium withMFL3 (10 μg/ml). Following embedding of the explants into collagen, cellinvasion was recorded over 72 hours with a time-lapse microscope(Olympus, Germany).

Example 5 Cells and Spheroid Cultures

The established glioblastoma cell lines A172, T98G and LN18 and theprimary glioblastoma cells (NCHs) were cultured in DMEM (supplementedwith 10% FCS and 1% PS) in a CO2 incubator at 36.5° C. and 90% humidity.The NCH cell lines have been established in the laboratory of C.Herold-Mende as described⁴⁸. For biochemical and molecular analysis1×10⁶ cells were plated onto 10 cm culture dishes in medium andincubated as described before. Spheroids were produced as previouslydescribed⁴⁹. In brief, T98G and LN18 cells (2-3×10⁴) were plated inhanging drops (20 μl) onto the lids of 10 cm culture dishes containing10 ml DMEM. After 48 h the cellular aggregates were harvested andtransferred onto a base-coated 2% agar dish filled with medium. Afteradditional 48 h spheroids were embedded in athree-dimensional-collagen-gel for invasion analysis.

Example 6 Collagen-Invasion-Assay

A physiological model for investigating invasion is thethree-dimensional-collagen-gel-assay. Spheroids were treated 1 h beforebeing embedded into a collagen gel solution (Vitrogen 3 mg/ml stocksolution; final concentration 2.4 mg/ml) with αApo-1 (2 μg/ml), LZ-CD95L(5 ng/ml) or Nok1 (50 ng)/LZ-CD95L (5 ng/ml). After polymerization ofthe collagen gel (3060 min at 37° C.) DMEM was put on top of the gels.The invasion of cells into the collagen matrix was documented with atime-lapse microscope (Olympus, Germany). The distance of singleinvading cells to the spheroid border (n=10 per spheroid; 3 spheroidsper treatment) was determined with Image J 1.34 software (based on NIHImage).

Example 7 Homogenization of Tissue

For FACS analysis, tumors were removed at indicated time points,trypsinized for 20 min at 37° C., washed thrice in PBS/10% FCS,triturated with glass Pasteur pipettes, filtered through a 100 μm nylonmesh (BD Falcon) and resuspended in PBS/10% FCS for FluorescenceActivated Cell Sorter (FACS, Becton Dickinson) analysis.

Example 8 Migration-Assay

Migration of the glioma cells in vitro was measured by the migrationthrough Collagen 1-coated (Chemicon) transwell inserts (Falcon). 5×10⁴cells were plated in 200 μl medium onto collagen-coated (50 μg/ml)transwell inserts with 8 μm pore size. Cells were either γ-irradiatedbefore plating or treated after plating with α-Apo1 (2 μg/ml or 0.1μg/ml+ProteinA for cross linking), LZ-CD95L (5 ng/ml), Nok1 (50ng)/LZ-CD95L (5 ng/ml), Nok1 (50 ng) LZ-Supernatant (SN, 5 ng/ml), CD95L(10 ng/ml) and CD95L-T34. Preferably 24 h after plating the cells werestarved with basal DMEM for additional 24 h before they were treated.The number of completely migrated cells was counted at 12 h, 24 h and 36h after treatment. In every experiment triplicates were counted for eachtreatment.

Example 9 Tumor Scoring and Analysis

Scoring of CD95L in original and recurrent gliomas was performed byanalysis of three areas (250-fold magnification) from each CD95L-stainedtumor. Positive cells were counted in a double blind manner and scoresassigned according to the number of positive cells.

For analysis of CD95-induced migration in vivo, a suspension of 5.000SMA-560 cells and 3 μg of MFL3 or the appropriate isotype antibody wasinjected into the left striatum of Vm/Dk mice. After one week, mice weresacrificed and the brains extracted. Following immunohistochemicalstaining, GFP positive cells in the contralateral hemisphere of threerepresentive areas per sample were counted and normalized to the surfaceof the tumor as assessed by Cell^R software (Olympus, Germany).

Example 11 γ-Irradiation

2.5×10⁵ cells were plated onto 6 cm culture dishes 12 h prior toirradiation. Cells were irradiated with 1, 3, 10 and 50 Gy at roomtemperature using a ¹³⁷Cs source (Gamma Cell 1000, Atomic Energy ofCanada, Ltd., ON) at 10.23 Gy/min. Cells were treated with Nok1 (10μg/ml) or left untreated directly after irradiation. Thereafter cellswere used for migration-assays, RNA extraction or Nicoletti-assay.

Example 12 Statistical Analysis

Statistical analysis of migration and mRNA expression data was performedusing the nonparametric Student t test to compare differences betweentreatment groups and controls. Confidence intervals were determined at95%, and *P values<0.05, **P value<0.01. ***P value<0.005 wereconsidered statistically significant.

Example 13 Immunoprecipitation

2×10⁷ cells were either treated with 10, 500 and 5 μg/ml CD95L-T4 for 1and 5 minutes (unless otherwise indicated) at 37° C. or left untreated,washed twice in PBS plus phosphatase inhibitors (NaF, NaN3, pNPP, NaPPi,β-Glycerolphosphate, 10 mM each and 2 mM orthovanadate), andsubsequently lysed in buffer A (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2mM EDTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail[Roche], 1% Triton X-100 [Serva, Heidelberg, Germany], 10% glycerol, andphosphatase inhibitors [NaF, NaN3, pNPP, NaPPi, β-Glycerolphosphate, 10mM each and 2 mM orthovanadate]) (stimulated condition) or lysed withouttreatment (unstimulated condition). Protein concentration was determinedusing BCA kit (Pierce). 1 mg of protein was immunoprecipitated overnightwith either anti-caspase-8 as previously described⁵⁸, 5 μg αApo-1 or 2.5μg anti-p85 and 30 μl protein-A Sepharose. Beads were washed 5 timeswith 20 volumes of lysis buffer. The immunoprecipitates were analyzed oneither 15% or 7.5% SDS-PAGE. Subsequently, the gels were transferred toHybond nitrocellulose membrane (Amersham Pharmacia Biotech, Freiburg,Germany), blocked with 2% BSA in PBS/Tween (PBS plus 0.05% Tween 20) for1 hour, and incubated with primary antibodies in 2% BSA PBS/Tween at 4°C. overnight. Blots were developed with a chemoluminescence methodfollowing the manufacturer's protocol (PerkinElmer Life Sciences,Rodgan, Germany).

Example 14 Cell Analysis by Flow Cytometry

Extracellular Staining:

Expression of CD95 on the surface of single cells was analyzed by FACS.1×10⁶ cells/ml in phosphate-buffered saline containing 10% fetal calfserum (PBS 10% FCS) were incubated with αApo-1 (0.01 μg/μl) for 20minutes on ice, followed by the secondary antibody (1:30 goat-anti-mousephycoerythrin-conjugated; Dianova) for 30 minutes. Flowcytometricanalysis was performed on a FACSCalibur (Becton Dickinson) using CellQuest Software. A minimum of 10.000 cells per sample was analyzed.

Intracellular FACS Staining:

To measure either the overexpression efficiency of PTEN, the knockdownefficiency of Yes or the basal levels of FADD, Fyn and Yes,intracellular FACS stainings were performed. Cells were trypsinized,supernatant was discarded and the pellet was resuspended in 4%paraformaldehyde in PB-buffer for 15 minutes on ice. After incubationthe fixed cells were centrifuged (3.000 rpm, 4° C., 5 minutes)supernatant was discarded and the pellet was washed twice with PBS/0.1%Saponin and 10% FCS. Samples were incubated on ice for 30 minutes withthe first antibody (α-HA 1:1000 Roche, α-Yes 1:200 or α-Fyn 1:200 SantaCruz), followed by two washing steps with PBS/0.1% Saponin and 10% FCSbefore addition of the secondary antibody (α-mouse-PE 1:33 Pharmingen orα-rabbit-Alexa488® 1:250 Molecular Probes) for additional 20 minutes.Stained cells were washed twice with PBS/0.1% Saponin and 10% FCS andthe pellet resuspended in the same buffer. Cells were then analyzed byFACS. Values were given as normalized mean fluorescent intensity (MFI)for the specific antigen.

Example 15 Detection of Apoptosis (Nicoletti Assay)

To quantify DNA fragmentation, cells detached with trypsin/EDTA (Gibco)were centrifuged at 200×g and fixed with 70% ethanol at −20° C. for 1 h.Fixed cells were stained with propidium iodide solution (50 μg/ml;0.0025% sodium citrate and 0.0025% Triton-X-100) for 1 h or overnight at4° C. and analyzed by FACS.

Example 16 Gelatin Zymography for MMP-2 and -9

Conditioned medium of untreated or treated (CD95L-T4 10 ng/ml and 20ng/ml) T98G cells were loaded under nonreducing conditions onto a 10%SDS-polyacrylamide gel containing 1 mg/mL gelatin. After electrophoresisand washing the gel with Triton X-100 (2.5% v/v, twice for 30 min), thegel was incubated in MMP reaction buffer [50 mmol/L Tris-HCl (pH 7.8),200 mmol/L NaCl, 5 mmol/L CaCl2] at 37° C. for 16 h. Gelatinolyticactivity was detected as transparent bands on staining with CoomassieBrilliant Blue G-250 solution and incubation in destaining solution (10%acetic acid, 20% methanol).

Example 17 Immunohistochemistry

T98G cells were fixed with 4% PFA at 37° C. for 15 minutes, incubatedwith 50 mM ammonium chloride and permeabilized with 0.1% Triton X-100 inPBS for 5 minutes. After blocking, cells were incubated with therespective primary antibodies, and immunoreactivities were visualizedwith a monoclonal or polyclonal antibody coupled to rhodamine orfluorescein isothiocyanate (FITC).

Clinical samples from GBM WHO IV were fixed with 4% PFA andparaffin-embedded. Consecutive sections of 5 μm thickness wereimmunostained with mouse antibodies against CD95L, Yes and phospho-Src(Tyr416). For validation of the anti-CD95L and anti-CD95 antibodies,human tonsils were used. After incubation with biotin-coupled secondaryantibodies followed by incubation with streptavidine-alkalinephosphatase (Dako) sections were developed with FastRed (Dako) andembedded with Glycergel (Dako).

Murine tumors were fixed with 4% PFA. After paraffin embedding,consecutive slices of 5 μm thickness were immunostained with rabbitanti-GFP.

Example 18 Lentivirus Infection

T98G and LN18 cells were infected with the lentiviral vector pEIGW andpEIGW-GSK3βS9A at a multiplicity of infection (MOI) of 5. The plasmidswere constructed by

replacing the eGFP sequence between the EF1a promotor and the WPREelement in pWPTSeGFP (kindly provided by D. Trono, Geneva) with theIRES-eGFP cassette from pIRES2-eGFP (Clontech, Germany). The recombinantlentiviral vector pEIGW-GSK3βS9A was constructed using pcDNA3HA-GSK3βS9A (kindly provided by Trevor C. Dale). This vector encodes aconstitutively active GSK3b mutant containing a serine-to-alaninesubstitution at residue 9 (GSK3βS9A). All lentiviruses were propagatedusing previously described methods⁴¹. Expression of all transgenes wasconfirmed in infected cells by FACS analysis of GFP expression. Thepercentage of infected cells was 80-90%.

Example 19 Real Time PCR

RNA from cells treated either with .alpha.Apo-1 (1 μg/ml) or leftuntreated was extracted with the Qiagen RNeasy Mini Kit at 48 h unlessotherwise stated. After the reverse transcription, target mRNA wasdetected by Taqman real-time PCR with the following gene-specificprimers: CD95-forw. 5′-ACT GTG ACC CTT GCA CCA AAT-3′ (SEQ ID NO:2);CD95-rev. 5′-GCC ACC CCA AGT TAG ATC TGG-3′ (SEQ ID NO:3); CD95-probe5′-AAT CAT CAA GGA ATG CAC ACT CAC CAG CA-3′ (SEQ ID NO:4); CD95L-forw.5′-AAA GTG GCC CAT TTA ACA GGC-3′(SEQ ID NO:5); CD95L-rev. 5′-AAA GCAGGA CAA TTC CAT AGG TG-3′ (SEQ ID NO:6); CD95L-probe 5′-TCC AAC TCA AGGTCC ATG CCT CTG G-3′ (SEQ ID NO:7); MMP-9-forw. 5′-GAT CCA AAA CTA CTCGGA AGA CTT G-3′ (SEQ ID NO:8); MMP-9-rev. 5′-GAA GGC GCG GGC AAA-3′(SEQ ID NO:9); MMP-9-probe 5′-CGC GGG CGG TGA TTG ACG AC-3′ (SEQ IDNO:10); MMP-2-forw. 5′-GGA CAC ACT AAA GAA GAT GCA GAA GT-3′ (SEQ IDNO:11); MMP-2-rev. 5′-CGC ATG GTC TCG ATG GTA TTC-3′ (SEQ ID NO:12);MMP-2-probe 5′-AGT GCC CCA GCA AGG TGA TCT TGA CC-3′ (SEQ ID NO:13);b-Actin-forw. 5′-ACC CAC ACT GTG CCC ATC TAC GA-3′ (SEQ ID NO:14);b-Actin-rev. 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′ (SEQ ID NO:15);b-Actinprobe 5′-ATG CCC TCC CCC ATG CCA TCC TGC GT-3′ (SEQ ID NO:16). Toproof the knockdown of Yes, target mRNA was detected by SybrGreenreal-time PCR with the use of the following primers of the Src kinasefamily: Yes-forw. 5′-TAT GGC TGC CAG ATT GCT G-3′ (SEQ ID NO:17);Yesrev. 5′-ZZC AGG AGC TGT CCA TTT GA-3′ (SEQ ID NO:18); Fyn-forw.5′-TGA ACA GCT CGG AAG GAG AT-3′ (SEQ ID NO:19); Fyn-rev. 5′-GGT TTV ACTCTC CGC GAT AA-3′ (SEQ ID NO:20); as housekeeping gene Gapdh was usedwith the following sequences: Gapdh-forw. 5′-GGT CGG AGT CAA CGG ATT TGGTCG-3′ (SEQ ID NO:21); Gapdh-rev. 5′-CCT CCG ACG CCT GCT TCA CCA C-3′(SEQ ID NO:22). The realtime PCR was measured in a ABIPRISM-7300i(Applied Biosystems, USA).

Example 20 PTEN Overexpression

Overexpression plasmids for PTEN (pBP-PTEN-HA) and the empty vector(pBP) were kindly provided by Frank Furnari (San Diego, USA)⁵⁹. T98Gcells were transfected with pBP-PTEN-HA and pBP (6 μg) using JetPei.Transfected cells were cultured for an additional 48-72 h beforetreatment.

Example 21 Knockdown Experiments

Knockdown experiments were performed by transient transfection withLipofectamine 2000™ (Invitrogen Life Technologies) following theinstruction manual. Migration experiments were performed using eithervalidated siRNA against Yes (Qiagen SI00302218), and a second siRNA,targeting a different member of the Src family kinases, Fyn (QiagenSI00605451), which was used as a negative control or pools of validatedshRNAmir-pGIPZvectors for Yes, MMP-2 and MMP-9 and a non-targetingshRNAmir-pGIZ vector as a negative control (RHS 4430-98843955,-98820654, -99161516, -98514235, -98709361, -99137419, -99291751,-99298712, -99138418 and RHS4346-OB respectively, Open Biosystems, USA).After transient transfection with the different siRNAs cells werecultured for 72 h before treated with CD95LT4 (10 ng/ml and 20 ng/ml),migration was analyzed 24 h after treatment with a two dimensionalmigration assay. The knockdown was controlled by quantitative real-timePCR and FACS. To exclude off-target effects of Yes-siRNA, cells weretransfected with siRNA against Yes, a Yes overexpression plasmid(p-CMV-Yes) or both and cultured for 48 h before being transferred to amigration plate. After additional 48 h cells were treated with CD95L-T4(10 ng/ml and 20 ng/ml). Migration was measured 24 h after treatment ina two dimensional migration assay.

For immunoprecipitation studies, transfected cells were cultured for 72h prior to treatment.

Example 22 Luciferase Reporter Gene Assays

Luciferase reporter vectors were kindly provided from the followingsources: pTOPFLASH and pFOPFLASH (Randall T. Moon, Howard Hughes MedicalInstitute of Washington; USA) and the NFκB plasmid with six NFκB-bindingsites (Min Li-Weber, German Cancer Research Center Heidelberg, Germany).Transfection experiments were carried out using Lipofectamine 2000™reagent (Invitrogen Life Technologies), according to the manufacturer'sinstructions. Cells were seeded in 24-well plates at a density of 5×104cells per well 24 h prior to transfection. The Firefly-Luciferaseconstructs were cotransfected with a CMV-Renilla-Luciferase plasmid (10ng) to normalize the luciferase values. Luciferase activity was measured24 h after transfection depending on the construct by using commerciallyavailable kits from Promega (Madison, Wis., USA). Luminescence wasquantified using a Ascient 96-well microplate luminometer. Alltransfections were carried out in quadruplicates on at least twoindependent occasions, and error bars represented as s.e.m.

Example 23 Generation of CD95L-T4

Genetic Engineering of Human CD95-Ligand-T4 (CD95L-T4)

The TRAIL/DR5 complex as well as the TNF-.alpha. structure were used asmodels to develop expression strategies for the human CD95L-receptorbinding domain (CD95L-RBD). Provided that the structure of trimerichuman CD95L-RBD is in principle similar to the TNF-.alpha.- orTRAIL-RBD-structures (PDB-entries: 1TNF and 1D0G/1DU3, respectively.sup.51, 55, 62), the following observations were taken into account:[0136] 1. The N- and C-terminal amino acids of the RBD from TRAIL andTNF-a form an antiparallel .beta.-strand. [0137] 2. The terminal aminoacids of this .beta.-strand are located next to each other at the samesite of the molecule close to the central axis of the TRAIL-RBD trimer(see FIG. 19). This means, that for steric reasons, the use of N- andC-termini in the same molecule for the fusion of protein domains (e.g.for the addition of stabilization motifs or tags) is mutually exclusive.The ideal stabilisation motif should be a small, well defined trimerlocated close to the central axis of the CD95L-trimer with its N- andC-terminus at opposite sites of the stabilisation motif in order tominimize its risk of interference with the ligand/receptor interactionsites. An appropriate trimeric protein domain fulfilling these criteriais the T4-Foldon motif from the fibritin of the bacteriophage T4.sup.61,65. According to the above mentioned considerations the T4-Foldon wasfused C-terminally to the human CD95L-RBD (Glu142-Leu281 of CD95L).Between the CD95L-RBD and the T4-Foldon, a flexible linker element(GSSGSSGSSGS; SEQ ID NO: 23) was placed and a hexahistidine tag and astreptag-II (HHHHH-HSAWSHPQFEK; SEQ ID NO:24) was added C-terminally.This affinity tag was linked to the T4-Foldon by a flexible linkerelement (SGPSSSSS; SEQ ID NO:25). To allow for secretory basedexpression, a signal peptide from human Igk was fused to the N-terminus(Glu142). The proposed signal peptide cleavage site formed by the fusionof the Iv leader to the CD95LRBD is expected to release a final productwith a N-terminal located Glutamine, corresponding to Glu142 of humanCD95L. The amino acid sequence of the CD95L-T4-construct shown in FIG.19C was backtranslated and its codon usage optimized for mammaliancell-based expression. Gene synthesis was done by ENTELECHON GmbH(Regensburg, Germany). The final expression cassette was subcloned intopCDNA4-HisMaxbackbone, using unique Hind-III- and Not-I-sites of theplasmid. A schematic summary, including all features described above, isshown exemplarily for the TRAIL-T4-DR5-complex (FIG. 19D).

Expression and Purification of CD95L-T4

Hek 293T cells grown in DMEM+GlutaMAX (GibCo) supplemented with 10% FBS,100 units/ml Penicillin and 100 μg/ml Streptomycin were transientlytransfected with a plasmid encoding CD95L-T4. Cell culture supernatantcontaining recombinant CD95L-T4 was harvested three days posttransfection and clarified by centrifugation at 300 g followed byfiltration through a 0.22 μm sterile filter. For affinity purification 1ml Streptactin Sepharose (IBA GmbH, Göttingen, Germany) was packed to acolumn and equilibrated with 15 ml buffer W (100 mM Tris-HCl, 150 mMNaCl pH 8.0). The cell culture supernatant was applied to the columnwith a flow rate of 4 ml/min. Subsequently, the column was washed withbuffer W and bound CD95L-T4 was eluted stepwise by addition of 7×1 mlbuffer E (100 mM Tris-HCl, 150 mM NaCl, 2.5 mM Desthiobiotin, pH 8.0).The protein content of the eluate fractions was analysed by SDS-PAGE andsilver staining (FIG. 19 E). Fractions E2-E5 were subsequentlyconcentrated by ultrafiltration and further analysed by size exclusionchromatography (SEC). SEC was performed on a Superdex 200 column usingan Äkta chromatography system (GEHealthcare). The column wasequilibrated with phosphate buffered saline and the concentrated,streptactin purified CD95L-T4 (E2-E5) was loaded onto the SEC column ata flow rate of 0.5 ml/min. The elution of CD95L-T4 was monitored byabsorbance at 280 nm. The apparent molecular weight of purified CD95L-T4was determined based on calibration of the Superdex 200 column with gelfiltration standard proteins (FIGS. 19 F and G) (Bio-Rad GmbH, München,Germany).

Apoptosis Assay

A cellular assay with a Jurkat A3 permanent human T-cell line (cat. no.CRL2570, ATCC) was used to determine the apoptosis inducing activity ofCD95L-T4. Jurkat cells were grown in flasks with RPMI1640-medium+GlutaMAX (GibCo) supplemented with 10% FBS (Biochrom), 100units/ml Penicillin and 100 μg/ml Streptomycin (GibCo). Prior to theassay, 100,000 cells were seeded per well into a 96-wellmicrotiterplate. The addition of different concentrations of CD95L-T4 tothe wells (final volume: 200 μl) was followed by a 3 h incubation at 37°C. Cells were lysed by adding 20 μl lysis buffer (250 mM HEPES, 50 mMMgCl₂, 10 mM EGTA, 5% Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) andplates were put on ice for 30 minutes. Apoptosis is paralleled by anincreased activity of caspase-3 and caspase-7. Hence, cleavage of thespecific caspase-3/-7 substrate Ac-DEVD-AFC (Biomol) was used todetermine the extent of apoptosis. In fact, Caspase activity correlateswith the percentage of apoptotic cells determined morphologically afterstaining the cells with propidium iodide and Hoechst-33342 (data notshown). For the Caspase activity assay, 20 μl cell lysate wastransferred to a black 96-well microtiterplate. After the addition of 80μl buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS, 50 μMAc-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to a TecanInfinite F500 microtiterplate reader and the increase in fluorescenceintensity was monitored (excitation wavelength 400 nm, emissionwavelength 505 nm) (FIG. 19 H).

This apoptosis assay was also used for the determination of biologicalactivity of the biopharmaceutical agent APG101. APG101—a fusion proteinof the extracellular domain of the human CD95-receptor (the in vivobinding partner of CD95 ligand) with human Fc—antagonizes the apoptosisinducing effect of CD95L. Prior to the addition of CD95L-T4 to theJurkat cells, CD95L-T4 at a constant concentration was incubated for 30minutes at 37° C. with different concentrations of APG101 (FIG. 19 I).

Example 24 CD95 Mediates Invasion of Glioblastoma Cells Resistant toApoptosis

In long-term human malignant glioma cell lines, we first examined theinduction of apoptosis upon triggering of CD95. Treatment with leucinezipper (LZ)-CD95L elicited variable effects in different glioma celllines: LZ-CD95L did not induce apoptosis in A172 cells, caused apoptosisonly at high doses in T98G cells or mediated apoptosis already at lowdoses in LN18 cells (FIG. 1a ). Specificity of LZ-CD95L-induced deathwas proven by the neutralization of apoptosis by an antibody to CD95L(NOK1; data not shown). The resistance of A172 to CD95-induced apoptosiscould be attributed to the low level of CD95 surface expression (FIG. 1b). LN18 and T98G cell lines, however, exhibited different sensitivity toapoptosis while showing comparably high levels of CD95 surfaceexpression (FIG. 1b ).

The potency to activate CD95 proportionally correlates with the degreeof oligomerization of CD95L. Since the available CD95L has a tendency toform aggregates, we engineered a human CD95L with a stable trimerbuilding capacity, the CD95L-T4 (FIG. 19). Different glioma cell linesexhibited different sensitivities to treatment with CD95L-T4: Apoptosiswas induced already at low concentrations in LN18 cells but not in T98Gcells (FIG. 11A). Specificity of CD95L-T4-induced death was tested bythe neutralization of apoptosis by an antibody to CD95L (NOK1; FIG. 21).Both LN18 and T98G cell lines, however, exhibited comparably high levelsof CD95 surface expression (FIG. 11A). These cell lines also expressedother molecules necessary for CD95-mediated apoptosis, such as FADD,caspase-8 or caspase-3 (FIGS. 15C and D)^(63,72).

Malignant glioma cells are characterized by their replicative potential,induction of angiogenesis, migration/invasion and evasion of apoptosis.Stimulation of CD95 did not alter the proliferation rate of T98G cells(data not shown). To test the invasion behavior we generated spheroidcultures of T98G and LN18 cells and plated them within a collagenmatrix. Treatment with LZ-CD95L increased invasion of migrating cellsinto the surrounding matrix to a higher extent in T98G than in LN18cells (FIG. 1c , FIG. 11 B). This was also the case when the cells wereplated in the upper chamber of two chambers separated by acollagen-coated membrane. The highly apoptosis-sensitive LN18 cells didnot react on CD95 activation with increase migration. T98G cells, incontrast, increased their migration potential upon treatment withLZ-CD95L or a stimulating antibody to CD95 (αAPO-1) (FIG. 3b ).

The migration of glioma cells requires cleavage of extracellular matrixcomponents through MMPs. In T98G cells, MMP-9 activity, as assessed bygel zymography, increased upon treatment with CD95L-T4 (FIG. 11C).Accordingly, stimulation of CD95 increased expression of MMP-2 and MMP-9mRNA levels in the migration-prone T98G but not in theapoptosis-sensitive LN18 cells (FIGS. 11D and E). Most importantly,CD95-induced migration of T98G cells could be blocked with siRNA poolagainst MMP-2 and -9, indicating that these MMPs are required forCD95-induced migration (FIGS. 1F and G).

In a further series of experiments we used short-term glioma culturesderived from patients' tumors. These cells exhibited the typicalGBM-genetic aberrations including single copy losses of the PTEN andCDKN2a loci and single copy gain of the EGFR locus, as assessed byarray-CGH analyses (Bernhard Radlwimmer, personal communication). Everyprimary GBM-derived culture examined here exhibited high CD95 surfaceexpression (n=18) and similar or higher levels of resistance toCD95-induced apoptosis (n=8) compared to the ones observed in theinvasion-prone T98G cell line (FIG. 12A, FIG. 20 and data not shown).Both the levels of CD95 surface expression and the resistance toCD95-mediated apoptosis were not affected by the number of passages inculture (data not shown). We further examined CD95-induced invasion inthe GBM-derived cultures NCH89, NCH125 and NCH270. Triggering of CD95 inNCH125 and NCH270 increased expression of MMP-2 and MMP-9 andsubsequently induced migration (FIG. 12B to D). Stimulation of CD95 inNCH89 cells neither increased migration nor expression of MMP-9 (FIG.12B to D). Thus, the migration response to CD95 does not strictlycorrelate with the degree of resistance to apoptosis. Along the sameline, expression of CD95 and CD95L mRNA differed among the highlyinvasive primary GBM tumors tested (FIG. 20). MMPs are required forCD95L-T4-induced migration of NCH125, as a siRNA pool to MMP-2 and -9significantly blocked migration (FIG. 12E).

Example 25 CD95 Mediates Invasion Via the PI3K/ILK/GSK/MMP Pathway in aCaspase-Independent Manner

The invasion of glioma cells requires cleavage of extracellular matrixcomponents through matrix metalloproteinases (MMP) as already outlinedabove. Accordingly, mRNA levels of MMP9 and MMP-2 greatly increased uponCD95 triggering in the migration-prone T98G but not in the apoptosisresistant LN18 cells (FIG. 2). The integrin-linked kinase (ILK) hasrecently been reported to mediate MMP9 expression via inhibition ofglycogen synthase kinase-3β (GSK3β)^(9,10). Inhibition of GSK3β viaphosphorylation at its serine-9 (phospho-ser9) residue was observed inT98G cells upon treatment with LZ-CD95L or αAPO-1 antibody (FIG. 3a andFIG. 8). Phosphorylation of GSK3β was also found in LN18 cells, but withdifferent kinetics (FIG. 3a and FIG. 8). The migration-prone T98G cellsexhibited higher basal phospho-ser9-GSK3β levels and graduallyincreasing long-lasting phosphorylation of GSK3β upon triggering of CD95(FIG. 3a and FIG. 8). The apoptosis-prone LN18 cells showed a transient(5-10 min) phosphorylation of GSK3β upon triggering of CD95 (FIG. 3a ).Overexpression of a constitutively active GSK3β mutant (GSK S9A) blockedCD95-induced migration of T98G cells (FIG. 3b ). GSK S9A-expressing T98Gcells and their wild-type counterparts exhibited comparable levels ofsensitivity to CD95-induced apoptosis and of growth rate (FIG. 3 c-d).Thus, inhibition of migration by constitutively active GSK3β in T98Gcells cannot be attributed to a different proliferation rate.Consequently, pre-treatment with the ILK inhibitor KP-SD-1 blockedCD95-mediated migration of T98G cells without affecting basal migration(FIG. 3e ). ILK activates protein kinase B (PKB/AKT) and inhibits GSK3βactivity in a phosphatidylinositol-3-kinase (PI3K)-dependent manner¹¹.Accordingly, inhibition of PI3K by LY294002 blocked CD95-induced AKTactivity and ser9 phosphorylation of GSK3β in T98G cells withoutchanging the phosphorylation status of the extracellular receptor kinase(ERK) (FIG. 3f ).

β-catenin forms a complex together with active GSK3β, the adenomatouspolyposis coli (APC) and axin proteins—the degradation complex.Phosphorylation of β-catenin by GSK3β targets it for proteasomaldegradation. As a consequence of GSK3β inhibition, β-catenin accumulatesand translocates into the nucleus where it engages the N-terminus ofDNA-binding proteins of the TCF/Lef family¹² inducing expression ofdifferent target-genes including MMPs. In T98G cells, triggering of CD95induced nuclear translocation of active β-catenin, not phosphorylated inthe GSK-targeted serine 37 or Threonine 41 (FIG. 3g ). Taken together,activation of CD95 induces migration/invasion through thePI3K/ILK/GSK3β/β-catenin/MMP pathway.

CD95 transduces the apoptotic signal through activation of caspases. Ithas recently been reported that CD95 mediates migration via activationof caspase-8, NFκB and ERK in mesenchymal tumor cells lines resistant toCD95-induced apoptosis¹³. In contrast to LN18 cells, CD95 stimulation ofT98G cells did not induce cleavage of caspase-8 (FIG. 3h ). Accordingly,pre-treatment of T98G cells with a broad-spectrum caspase inhibitor,benzoyl-VAD.fluoromethyl ketone (zVAD.fmk) did not block ser9phosphorylation of GSK3β in T98G cells (FIG. 8). Pre-treatment of T98Gcells with the MEK inhibitor PD98059 also did not interfere withCD95-induced migration (FIG. 3i ).

In addition to caspases, the phosphoprotein enriched indiabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15) has aDED and can, therefore, interact with other molecules at the DISC.Overexpression of PED has been reported to block CD95- andTNFR-1-induced apoptosis through simultaneous activation of ERK andinhibition of Jun N-terminal kinase (JNK)^(14,15). The anti-apoptoticactivity of PED increases if phosphorylated by AKT¹⁶. In T98G cells,short interference (si)-RNA to PED but not a control siRNA decreased PEDlevels and its reported activation of ERK, but not the CD95-mediatedinactivation of GSK3β (FIG. 8). In addition the levels of FLIPL, anothermolecule that can be recruited to the DISC and inhibit apoptosis,remained unaffected upon treatment with LZCD95L (data not shown).

One of the best described inducers of GBM invasion is EGF. Its bindingto EGFR promotes MMP-9 expression through activation of the MAPK/ERK andthe PI3K pathway²⁷. PI3K activates AKT/PKB, which in turn is able tophosphorylate GSK3β leading to its inactivation. To test if PI3K or MAPKsignalling could be responsible for the observed invasion we determinedphosphorylation of ERK and AKT. Stimulation of T98G and LN18 cells withCD95L-T4 activated AKT but not ERK (FIG. 13A). Interestingly, ERKactivity was even blocked with increasing time following stimulation(FIG. 13A). In the invasion prone T98G, NCH125 and NCH270 cells,phosphorylation of AKT exhibited a concentration dependent bell-shapedcurve (FIG. 13B). In contrast, in NCH89 cells, which did not react toCD95 with increased invasion, CD95L-T4 did not activate AKT above basallevels (FIG. 13B). Inhibition of GSK3β via phosphorylation at itsserine-9 (phospho-ser9) was observed in T98G cells upon treatment withCD95L-T4 or αApo-1 antibody by Western Blot and FACS staining (FIG. 13Cand FIG. 21).

Overexpression of a constitutively active GSK3β mutant (GSK S9A) vialentiviral infection blocked CD95-induced migration of T98G cells (FIG.13D). GSK S9A-expressing T98G cells and their wild-type counterpartsexhibited comparable growth rate and levels of sensitivity toCD95-induced apoptosis (FIG. 21). Thus, inhibition of migration byconstitutively active GSK3β in T98G cells cannot be attributed to adifferent proliferation rate. Active GSK3β forms a complex withβ-catenin, the adenomatous polyposis coli (APC) and axin proteins—thedegradation complex. Phosphorylation of β-catenin by GSK3β targets itfor proteasomal degradation. As a consequence of GSK3β inhibition,β-catenin accumulates and translocates into the nucleus, where itengages the N-terminus of DNA-binding proteins of the TCF (T-cellfactor)/Lef (lymphoid enhancing factor) family¹², inducing expression ofdifferent target genes including c-Jun, an essential transcriptionfactor for MMP-9 expression^(31,32). Alternatively, inhibition of GSK3βactivity can directly increase AP-1 expression¹⁰. To study whetherstimulation of CD95 triggers β-catenin's transcriptional activity weexamined expression of cytoplasmic and nuclear β-catenin and β-catenin'stranscriptional reporter activity. LiCl, a known inhibitor of GSK3β andinducer of β-catenin's transcriptional activity was used as a positivecontrol. In T98G cells, triggering of CD95 induced cytoplasmicaccumulation of β-catenin 30 minutes after stimulation with CD95L-T4(FIG. 13E). Further, nuclear translocation of active β-catenin,non-phosphorylated on the GSK targeted Ser 37 or Thr 41 was observed(FIG. 13F). TCF/Lef-reporter activity (TOP-FLASH) was also significantlyinduced upon CD95L-T4 (FIG. 13G). Mutation of the TCF/Lef binding domainabolished CD95L-T4 induction of luciferase activity (FOP-FLASH; FIG.13G). Additionally, activity of NFκB increased significantly 8 h afterstimulation with 20 but not 10 ng/ml CD95L-T4 (FIG. 13H). Takentogether, activation of CD95 induces migration/invasion through thePI3K/AKT/GSK3β/β-catenin/MMP and possibly the PI3K/AKT/NFκB/MMP pathway.

Example 26 CD95-Induced Migration is Also Detected in Primary GliomaCultures Resistant to Apoptosis

In a further series of experiments we used short term glioma culturesderived from patients' tumors. Cells from diffuse astrocytoma (WHO II)exhibited high CD95 surface expression and were relatively sensitive toCD95-mediated apoptosis (FIG. 4). In contrast, cells originating fromoligodendroglioma (WHO III) or glioblastoma (WHO IV) were highlyresistant to CD95-mediated apoptosis despite high CD95 surfaceexpression (FIG. 4 and FIG. 9). Every primary GBM-derived cultureexamined here exhibited high CD95 surface expression (n=18) and similaror higher levels of resistance to CD95-induced apoptosis (n=8) incomparison to the ones observed in the invasion-prone T98G cell line(FIG. 9). Both the levels of CD95 surface expression and the resistanceto CD95-mediated apoptosis were not affected by the number of passagesin culture (data not shown). We further tested three GBM-derivedcultures which were relatively (NCH125) or highly resistant (NCH89 andNCH270) to CD95-induced apoptosis. Triggering of CD95 in NCH125 andNCH270 increased expression of MMP9 and MMP-2 and subsequently migration(FIG. 5a-c ). Stimulation of CD95 in NCH89 cells neither increasedmigration nor expression of MMP9 and MMP-2 (FIG. 5b-c ). Thus, themigration response to CD95 does not strictly correlate with the degreeof resistance to apoptosis. Along the same line, expression of CD95 andCD95L mRNA was very different among the highly invasive primary GBMtumors tested (FIG. 9).

Example 27 Irradiation Increases Invasiveness Via the CD95/CD95L System

In the clinical setting, invading cells that escape surgery are thetargets of radiotherapy and adjuvant chemotherapy. γ-irradiation hasbeen reported to increase expression of CD95 and CD95L and therebyinduce apoptosis³. Considering our present data we wanted to addresswhether irradiation-induced CD95 and CD95L could also increaseinvasiveness of glioma cells. First, we showed that irradiation of T98Gcells increases expression of CD95 and CD95L mRNA (FIG. 6a ). Thehighest expression of CD95 and CD95L mRNA was found at a dose of 3 Grays(Gy). At the same dose, MMP-2 mRNA was significantly induced (FIG. 6b ).MMP9 mRNA was also significantly upregulated at 3 and 10 Gy but to alower extent (FIG. 6b ). Most importantly, MMP expression was mirroredby a higher migration rate of irradiated cells that could be reverted byneutralization of CD95L (FIG. 6c ). Primary GBM cultures also exhibiteda more invasive phenotype following irradiation (FIG. 6d and FIG. 10).Irradiation-induced migration was fully CD95L-dependent (FIG. 6d ).Interestingly, even in NCH89 cultures that did not exhibit an invasivephenotype after direct triggering of CD95, 10 Gy irradiation increasedthe number of migrating cells via CD95L (FIG. 6d ). Irradiationsignificantly induced migration in a CD95-dependent manner in nine outof the ten GBM-derived primary cultures examined here (FIG. 6d and FIG.10). The only culture that failed to exhibit a significant tendency tomigrate upon CD95 stimulation had lower CD95 surface expression levels(NCH 417; FIG. 10). We further studied expression of these molecules inrecurrent-tumors arising after surgery and irradiation of the originaltumor. Expression levels of CD95L within the tumor were scored from 0 to4 (FIG. 6e ). While levels in the first detected glioma were never above0 (1-24 CD95L-positive cells per field), a dramatic increase of CD95Lexpression following radiotherapy was detected in eight of the ninerecurrent tumors studied (FIG. 6e ). CD95L was detected in GFAP-positivetumor cells (FIG. 6f ). Additional expression of CD95 and MMP9 wasdetected in the same region in consecutive slices (FIG. 6f ).Importantly, apoptotic cells were not observed near CD95L-expressingcells (data not shown).

Example 28 PI3K is Activated Via Recruitment of Src to CD95

Src connects CD95 to PI3K activity as shown by co-immunoprecipitationexperiments (FIG. 14A to C). Indeed, treatment of T98G and LN18 cellswith CD95L-T4 induced recruitment of Src and the p85 subunit of PI3K toCD95. Association of p85 with CD95 was examined by immunoprecipitatingeither CD95 or p85. The degree of association of p85 with CD95 inverselycorrelated with the concentration of CD95L-T4 in T98G cells (FIG. 14B).However, in LN18 cells p85 recruitment to CD95 was only detected at highconcentrations of CD95L-T4 (FIG. 14A). Immunoprecipitation of CD95allowed detection of a Src-family member at five minutes after treatmentwith low concentration of CD95L-T4 (FIGS. 14A and B). Src associationdecreased at a higher concentration (FIGS. 14A and B). Thus, at lowconcentrations of CD95L-T4 both Src and p85 associated at detectablelevels with CD95 in T98G cells but in LN18 cells only Src was detected.Further, after a screening with antibodies to several SFKs, such as Fyn,Lyn, pp60 and Yes, we identified Yes as the Src-family member whichlinks CD95 to PI3K (FIG. 14C). To validate the role of Yes in themigration of glioma cells, knockdown experiments were performed. Incells transfected with Yes siRNA, expression of Yes, as assessed by FACSand qRT-PCR, was reduced while Fyn expression, another Src-familymember, remained unaffected (FIG. 14E). siRNA to Yes but not to Fyn,significantly abolished CD95L-T4-induced migration of T98G and of NCH125cells (FIG. 14D). This block of migration was rescued by Yesoverexpression in T98G and LN18 cells (FIG. 14F). Like the PI3Kinhibitor LY290059, siRNA to Yes also inhibited CD95-inducedphosphorylation of AKT (FIG. 14G).

Example 29 Inefficient DISC Formation in Apoptosis Resistant GliomaCells

The role of the PI3K pathway repressor PTEN (MMAC1, TEP1) was examined.While the apoptosis prone LN18 cells have an intact PTEN, T98G cellscarry a point mutation (codon 42 CTT to CGT; Glycine to Glutamine) inone allele and lack of the second allele of PTEN and a total loss of oneof the chromosome 10^(57,59). PTEN overexpression, however, did notsensitize T98G nor NCH125 cells to CD95-mediated apoptosis (FIG. 15A).

We further questioned whether caspases were involved in CD95-inducedactivation of PI3K. Inhibition of caspases by the general caspaseinhibitor zVAD-fmk did not prevent GSK3β phosphorylation (FIG. 15B).Likewise, CD95-induced cleavage of caspase-8 could only be detected inLN18 but not in T98G cells (FIG. 15C). To investigate if DISC componentswere efficiently recruited in these cells we analyzed FADD recruitmentin CD95-immunoprecipitates. Whereas upon stimulation with CD95L-T4recruitment of FADD to CD95 increased in LN18 cells, no increase wasdetected in T98G cells (FIG. 15D). Accordingly, caspase-8 recruitment toCD95 increased upon stimulation with CD95L-T4 in LN18 and J16 cells butnot in T98G cells (FIG. 15D). Most importantly, in T98G cells, siRNAknockdown of Yes enabled CD95L-T4 induction of FADD recruitment to CD95(FIG. 15E). Along this line, while expression levels of FADD weresimilar in LN18 and T98G cells, Yes levels were significantly higher inT98G cells (FIG. 15F). As opposed to Yes, Fyn expression wassignificantly higher in LN18 cells (FIG. 15F).

Example 30 The CD95/CD95L System is an Important Mediator of GliomaInvasion In Vivo

Expression of CD95L in patients suffering from Glioblastoma multiformeshowed a triangle-like distribution of CD95L in every tumor examined(FIG. 7a , 17A). Inside the tumor, only small amounts of CD95L wereexpressed (FIG. 7a .1, 17A.a). Expression increased at thetumor-parenchyma interface (FIG. 7a .2), peaked in the brain parenchymaadjacent to the tumor (FIG. 7a .3, 17A.b) and decreased again withincreasing distance to the glioma (FIG. 7a .4, 17A.c). CD95L wasdetected in glioma cells, neurons and macrophages (data not shown).Additional expression of CD95L within the tumor was observed in gliomacells surrounding tumor vessels. Likewise, phosphorylation of Src familykinases (pSrc) and Yes expression were consistently found at thetumor-host interface in every examined sample, suggesting a role intumor invasion (FIG. 17B). Within solid tumor areas, expression of Yeshighly varied between tumor samples, from very high to expression onlyin scattered tumor cells. In this highly Yes-expressing areasphosphorylation of Src was either not detected or rather limited (FIG.17B).

For translation of our findings into a more physiological in vivosetting we examined the role of the CD95/CD95L system in a mouse modelof Glioblastoma multiforme. For these studies, the established murineglioma cell line SMA-560 was injected intracranially into a syngenicVm/Dk host as described. The use of a syngenic tumor model was importantto allow tumor's induction of CD95L expression in surrounding braintissue.

SMA-560 cells expressed only low levels of CD95 receptor on theirsurface (FIG. 7b , 18A) and no CD95L at all (FIG. 7c , 18B) when keptunder cell culture conditions. As reported by others¹⁷ we found SMA-560cells to be resistant to CD95-induced apoptosis (Data not shown).Following the formation of spheroids, the levels of CD95 slightlyincreased (FIG. 7b , 18A), whereas FACS analysis failed to identifyCD95L at the cell surface (FIG. 7c , 18B). Despite the relatively lowamount of CD95 surface levels, spheroids formed from these cells displayincreased migration in the collagen invasion assay after CD95stimulation in a dose-dependant manner (FIG. 7d , 18C). In accordancewith our finding that spheroids do not express CD95L (FIG. 7c , 18B),blockage of CD95L using the CD95L neutralizing antibody MFL3 did notalter invasion (FIG. 7d , 18C).

Interestingly, FACS analysis of the surface levels of CD95 and CD95Lshowed a significant increase of both molecules (FIGS. 7b and 7c , 18Aand 18B) when cells isolated from solid tumors were analysed 14 and 18days after inoculation. This indicates the requirement of tumor-hostinteraction and, therefore, a cross-talk between host factors and tumorcells as given in the case of murine GBM.

For a more detailed analysis of the functional significance of thisincrease, we extracted fragments from solid tumors 14 days afterintracranial injection of cells and preincubated these for one hour witheither medium alone, medium with MFL3 or the appropriate isotypeantibody, respectively. After embedding into collagen gels, migrationwas monitored for a period of 72 hours (FIG. 7e , 18D). Strikingly,preincubation with the CD95L neutralizing antibody MFL3, but not withthe isotype or medium alone, reduced migration of cells out of the tumorcore by approximately 50% (FIG. 7e , 18D).

To verify these results in vivo, we injected GFP-positive SMA-560 cellsand MFL3 or the appropriate isotype antibody into the left striatum ofVm/Dk. Treatment of mice with MFL3 significantly reduced migration oftumor cells into the contralateral hemisphere (FIG. 7f , 18E) Weconclude from these data that the CD95/CD95L system is a major mediatorof malignant glioma invasion into the surrounding brain in vivo.

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What is claimed is:
 1. A method for treating high grade glioma in anindividual, comprising administering to an individual having high gradeglioma the only active ingredient of an anti-human CD95 ligand (CD95L)antibody or an antigen binding fragment thereof that neutralizes CD95Lactivity, wherein the only active ingredient is administered in anamount sufficient to reduce glial cell migration and/or invasion.
 2. Themethod of claim 1, wherein the only active ingredient is an antibodythat binds to human CD95L.
 3. The method of claim 2, wherein theantibody is Nok1.
 4. The method of claim 1, wherein the high gradeglioma is a WHO Grade III or IV glioma.
 5. The method of claim 1,wherein the high grade glioma is a WHO Grade IV glioma.
 6. The method ofclaim 1, wherein the only active ingredient is administered in theamount to reduce the glial cell migration to invade the contralateralhemisphere.
 7. The method of claim 1, wherein the high grade glioma isglioblastoma multiforme.